PCV2 replicase transcripts in infected porcine kidney (PK15) cells.

The Circoviridae family includes small viruses containing circular single strand DNA. There are two circoviruses described in pigs. Porcine Circovirus 2 (PCV2) has been linked to a disease termed Postweaning Multisystemic Wasting Syndrome (PMWS) whereas a close relative of this virus, termed PCV type 1 (PCV1) has been considered a non-pathogenic contaminant of the PK15 cell line. Rolling circle replication of small DNA viruses like circoviruses is mediated by a replicase (Rep) gene that is transcribed into different mRNAs by an alternative splicing mechanism. In PCV1, two transcripts Rep and Rep' implicated in DNA replication, have been identified from infected cells. These transcripts are the product of a single gene with Rep' having a different carboxyl end than Rep. Sequence comparison shows that there is a high degree of homology for the Rep gene in both viruses. In this article, we have identified three different transcripts by RT-PCR in PCV2 infected PK15 cells. Two of them have homology with their PCV1 counterparts and the third transcript, termed Rep", corresponds to another spliced version of the Rep gene. This transcript, unique to PCV2, encodes for a protein of 80 aminoacids in frame with the Rep sequence.

Representational differential analysis detects amplification of satellite sequences in postweaning multisystemic wasting syndrome of pigs.

Representational difference analysis (RDA) was used as a molecular approach to identify unique sequences associated with postweaning multisystemic wasting syndrome (PMWS) in pigs. Three rounds of subtractive hybridization and amplification between driver DNA extracted from normal pigs and tester DNA from PMWS-affected animals were performed. The final product corresponding to sequences associated with PMWS in pigs was analyzed using agarose gel electrophoresis, and 9 fragments were visualized after staining with ethidium bromide. Eight recombinants were successively cloned and sequenced, and the results were then compared with existing databases. Most of the PMWS clones isolated were satellite sequences from pig centrometric regions and 1 was a microsatellite sequence. One clone represented a microsatellite sequence, and 2 clones showed no homology with any gene found in the databases. The sequence comparison data did not reveal any homology with an infectious agent such as a virus or a bacterium. In the present experimental setting, it was concluded that PMWS in pigs triggers molecular changes such as an amplification of genomic regions containing repeated sequences.

Coinfection by porcine circoviruses and porcine parvovirus in pigs with naturally acquired postweaning multisystemic wasting syndrome.

Postweaning multisystemic wasting syndrome (PMWS) is an emerging disease in swine. Recently, the disease has been reproduced with inocula containing a newly described porcine circovirus (PCV), designated PCV 2, and porcine parvovirus (PPV). In order to determine if these viruses interact in naturally acquired PMWS, affected tissues from field cases were examined by immunohistochemistry (IHC) and polymerase chain reaction (PCR) for PCV 2 and PPV, as well as by PCR for the other recognized porcine circovirus, PCV 1. Porcine circovirus 2 was detected by PCR or IHC in affected fixed or frozen tissues from 69 of 69 cases of PMWS collected over 3 years from 25 farms. Porcine parvovirus was detected in 12 of the same cases, and PCV 1 was detected in 9 of 69; however, an apparent decrease was found in the sensitivity of the PCRs used to detect the latter 2 viruses when fixed tissue from the same cases were compared with the use of frozen tissues. Porcine circovirus 2 was not detected by PCR in affected tissues from 16 age-matched pigs that had Streptococcus suis-associated disease. Electron microscopic examination of plasma pooled from 15 pigs with PMWS revealed the presence of PCV and PPV, whereas these viruses were not observed in pooled plasma from 5 age-matched clinically normal pigs. These results confirm and extend previous findings documenting a consistent association of PCV 2 with PMWS. As well, infection by PPV or PCV 1 or both may be an important cofactor in the pathogenesis of some, but apparently not all, cases of PMWS.

Lack of evidence of conserved lentiviral sequences in pigs with post weaning multisystemic wasting syndrome.

In order to investigate the role of retroviruses in the recently described porcine postweaning multisystemic wasting syndrome (PMWS) serum and leukocytes were screened for reverse transcriptase (RT) activity, and tissues were examined for the presence of conserved lentiviral sequences using degenerate primers in a polymerase chain reaction (PCR). Serum and stimulated leukocytes from the blood and lymph nodes from pigs with PMWS, as well as from control pigs had RT activity that was detected by the sensitive Amp-RT assay. A 257-bp fragment was amplified from DNA from the blood and bone marrow of pigs with PMWS. This fragment was identical in size to conserved lentiviral sequences that were amplified from plasmids containing DNA from several lentiviruses. Cloning and sequencing of the fragment from affected pigs, however, did not reveal homology with the recognized lentiviruses. Together the results of these analyses suggest that the RT activity present in tissues from control and affected pigs is the result of endogenous retrovirus expression, and that a lentivirus is not a primary pathogen in PMWS.

Reproduction of lesions of postweaning multisystemic wasting syndrome in gnotobiotic piglets.

Neonatal gnotobiotic piglets were inoculated with tissue homogenates and low- and high-passage cell culture material to determine if the lesions of the newly described porcine postweaning multisystemic wasting syndrome (PMWS) could be reproduced. For this, 17 3-day-old gnotobiotic piglets were inoculated intranasally with pelleted chloroform-treated, filtered extracts from cell cultures, filter-sterilized homogenates of lymphoid tissue from PMWS-affected piglets, or control materials. Piglets were maintained in germ-free isolators for up to 5 weeks after infection prior to euthanasia and collection of samples for analysis. All piglets inoculated with the viral inocula developed lesions typical of PMWS, including generalized lymphadenopathy, hepatitis, nephritis, interstitial pneumonia, myocarditis, and gastritis. Porcine circovirus (PCV), as well as porcine parvovirus (PPV), was detected in tissues by virus reisolation, polymerase chain reaction analysis, or immunohistochemistry. All infected piglets developed moderate to high titers of antibody to PCV and moderate titers to PPV. No lesions, virus, or virus-specific antibodies were detected in sham-inoculated or uninoculated control piglets. These studies demonstrate that the lesions of PMWS can be experimentally reproduced in gnotobiotic piglets using filterable viral agents derived from pigs with PMWS and provide an experimental basis for further investigation into the pathogenesis and control of this emerging infectious disease in swine.

HIV-1 tat molecular diversity and induction of TNF-alpha: implications for HIV-induced neurological disease.

Activation and infection by HIV-1 of glial cells and infiltrating macrophages are cardinal features of AIDS-related neurological disease. Tumor necrosis factor-alpha (TNF-alpha) is released by these cell types, and increased TNF-alpha mRNA and protein levels are associated with the development and severity of HIV-induced neurological disease. HIV-1 proteins have been implicated in HIV neuropathogenesis including Tat which has been shown to be a potent inducer of TNF-alpha. We review our data showing the induction of TNF-alpha by Tat in primary human fetal astrocytes, human peripheral blood mononuclear cells, macrophages, and astrocytic and macrophage cell lines. TNF-alpha induction was NF-kappaB dependent and was eliminated by inhibiting protein kinase A, phospholipase C and protein tyrosine kinase activity. In addition, we examined the molecular diversity of the tat genome in the brains of HIV-infected patients from different HIV-1 clades. Comparison of matched brain- and spleen-derived tat sequences indicated that homology among brain-derived clones was greater than that between the brain- and spleen-derived clones. The brain-derived tat sequences were markedly heterogeneous in regions which influence viral replication and intracellular transport. Future studies using Tat, encoded by different sequences, will be necessary to determine the functional significance of tat molecular diversity. Nonetheless, these studies suggest that Tat is an important inducer of TNF-alpha production and thus may play a key role in the pathogenesis of HIV-related neurological disease.

Brain-derived HIV-1 tat sequences from AIDS patients with dementia show increased molecular heterogeneity.

HIV-1 infection results in a dementing illness affecting 20% of patients with AIDS. Several HIV-1 genes have been implicated in the pathogenesis of HIV-induced neurological disease. To search for distinct HIV-1 sequences associated with the development of dementia, brain-derived tat, env, and pol sequences were examined from AIDS patients defined pre-mortem as demented (HIV-D)[n=5] or non-demented (HIV-ND)[n=5]. Estimations of evolutionary distances and frequency of non-synonymous mutation rates revealed significant differences between brain-derived tat, env, and pol-encoded reverse transcriptase sequences. However, established zidovudine-associated resistance mutations in reverse transcriptase sequences were identified in only one HIV-D and one HIV-ND patient despite prolonged treatment of some patients. Non-synonymous/synonymous substitution rates among the tat sequences derived from patients with HIV-D were significantly higher compared to the HIV-ND group (P < 0.001). The ratios of transversions to transitions were also significantly higher among the HIV-D tat sequences (P< 0.01). Phylogenetic analyses showed clustering of sequences from each clinical group among the brain-derived tat and env sequences. These studies indicated that differing selective forces act on individual HIV-1 genes in the brain which may influence the development of dementia.

Protein and DNA elements involved in transactivation of the promoter of the bovine herpesvirus (BHV) 1 IE-1 transcription unit by the BHV alpha gene trans-inducing factor.

In herpes simplex virus (HSV)-infected cells, the transcription of immediate-early (alpha) genes is regulated by a virion component, the alpha gene trans-inducing factor (alpha TIF). This protein forms a complex with cellular factors and TAATGARAT motifs present in one or more copies in the promoters of all alpha genes. We have characterized the bovine herpesvirus 1 (BHV-1) homolog of this protein. Like its HSV counterpart, the BHV alpha TIF was synthesized in the later stages of infection and could be demonstrated to be a component of purified virions. In transient expression assays, BHV alpha TIF was a strong transactivator and stimulated the activity of IE-1, the major BHV-1 alpha gene promoter, with an efficiency comparable to that of HSV alpha TIF. This stimulation was largely dependent on a TAATGAGCT sequence present in a single copy in IE-1, and BHV alpha TIF, in conjunction with cellular factors, formed a complex with oligonucleotides containing this sequence. Despite these similarities between the two alpha TIFs, our preliminary observations suggest that the proteins may activate transcription by different mechanisms. Although BHV alpha TIF strongly transactivated IE-1, it differed from its HSV counterpart in that the carboxyl terminus of BHV alpha TIF, when fused to the DNA-binding domain of GAL4, was a relatively poor stimulator of a promoter containing GAL4-binding sites. Also unlike HSV alpha TIF, removal of the carboxyl terminus of BHV alpha TIF reduced but did not eliminate the ability of the protein to transactivate IE-1. These results are discussed in view of the structural similarities and differences among the alpha TIFs of alphaherpes-viruses.

The latency-related gene of bovine herpesvirus 1 inhibits the activity of immediate-early transcription unit 1.

Bovine herpesvirus 1 (BHV-1) establishes a latent infection in sensory neurons of infected animals. Only one virus-encoded latency-related (LR) gene is expressed during a latent infection. The LR transcript overlaps immediate-early transcription unit 1 (IEtu1) and is anti-sense with respect to IEtu1. The transcriptional start site of the LR RNA was mapped to position 724 of the LR gene, downstream from two putative TATA elements. When LR promoter sequences were deleted from a plasmid containing IEtu1 and the LR gene, the resulting construct trans-activated the HSV-1 thymidine kinase (TK) promoter more efficiently than IEtu1 plus the LR gene. Cotransfection of a plasmid containing the intact LR gene with IEtu1 inhibited the ability of IEtu1 to trans-activate the TK promoter. These results imply that a LR gene product(s) repressed the trans-acting capacity of IEtu1.

Localization of cis-acting sequences in the latency-related promoter of bovine herpesvirus 1 which are regulated by neuronal cell type factors and immediate-early genes.

Bovine herpesvirus 1 (BHV-1) establishes a latent infection in sensory ganglionic neurons of cattle. During a latent infection, a single latency-related (LR) transcript is expressed. This observation suggested that DNA sequences in the LR promoter are positively regulated by neural cell type factors. The regulation of the LR gene was examined in neural cells as well as nonneural cells in transient assays. A 258-bp XbaI-SphI fragment from the LR promoter cis activated the herpes simplex virus type 1 thymidine kinase promoter in rat pheochromocytoma (PC-12) cells and differentiated human (HCN1A) neurons. In contrast, cis activation was not observed with rat (Rat-2) fibroblasts, undifferentiated HCN1A cells, or bovine turbinate cells. Treatment of PC-12 cells with nerve growth factor increased transcriptional activity of the XbaI-SphI fragment. Exonuclease III footprinting experiments suggested that nuclear factors bind to the XbaI-SphI fragment. The immediate-early genes of BHV-1 trans activated the LR promoter, and DNA sequences 5' to the XbaI-SphI fragment were necessary for maximal stimulation. These results imply that neural-cell-type-specific factors and BHV-1 immediate-early genes positively regulate LR gene expression.

Comparative studies of BHV-1 variants by in vivo--in vitro tests.

The new encephalitogenic BHV-1.3 and previously characterized BHV-1 strains were studied with reference to their immunogenic and protective potency and their antigenic relationships using "in vitro" and "in vivo" tests. The "in vitro" results obtained by neutralization kinetics showed that the Los Angeles (LA) strain (BHV-1.1) and a vaginal isolate L-114 strain (BHV-1.2) had antigenic similarities. Conversely, the behavior of the encephalitogenic strain A-663 (BHV-1.3), was significantly distinct. The "in vivo" protection test was carried out in calves using LA and A-663 strains. Post-vaccination antibodies and challenge with A-663 strain showed that the immunogenic behavior and protective capacities of both strains were similar. Neutralization kinetics differences between BHV-1.1 and BHV-1.3 did not alter the "in vivo" protection against BHV-1.3 challenge.

Comparison of three serological techniques for the diagnosis of bovine herpesvirus type 1: serum neutralization, enzyme-linked immunosorbent assay and indirect immunofluorescence

La seroneutralización (SN) en cultivos celulares, hemoaglutinación pasiva (HA), la inmunofluorescencia indirecta (IFI) y las pruebas inmunoenzimáticas (ELISA) son de utilizadas para la detección de anticuerpos específicos contra Herpesvirus-bovino 1 (BHV-1). La prueba de SN es la determinación de referencia, sin embargo las dificuldades de implementación por la necessidad de contar con cultivos celulares, así como la celeridad en la obtención de resultados, determinan la optimización de otra prueba de alternativa y equivalente especificidad y sensibilidad. Trabajos previous describen una alta correlación entre las técnicas de SN vs IFI y de SN vs ELISA, destacando una mayor sensibilidad para el ELISA. El presente trabajo compara la sensibilidad y especificidad de IFI, ELISA y SN en la detección de anticuerpos a BHV-1 utilizando 105 sueros bovinos. La especificidad de las técnicas comparadas se demostró por la seroconversión obtenida en sueros de animales experimentalmente infectados (Cuadro 1). Se observó una alta asociación entre sueros reactivos y no reactivos entre SN e IFI, SN y ELISA y entre Ifi y ELISA destacándose una mayor sensibilidad para el ELISA (Figuras 2 y 3). El estudio estadístico de los resultados obtenidos con las tres técnicas diagnósticas determinó un alto coeficiente de correlación entre las mismas (Cuadro 2). Debido a la simplicidad y facilidad de ejecución se recomienda la técnica de ELISA para estudios epizootiológicos de larga escala (AU)

Comparison of three serological techniques for the diagnosis of bovine herpesvirus type 1: serum neutralization, enzyme-linked immunosorbent assay and indirect immunofluorescence

La seroneutralización (SN) en cultivos celulares, hemoaglutinación pasiva (HA), la inmunofluorescencia indirecta (IFI) y las pruebas inmunoenzimáticas (ELISA) son de utilizadas para la detección de anticuerpos específicos contra Herpesvirus-bovino 1 (BHV-1). La prueba de SN es la determinación de referencia, sin embargo las dificuldades de implementación por la necessidad de contar con cultivos celulares, así como la celeridad en la obtención de resultados, determinan la optimización de otra prueba de alternativa y equivalente especificidad y sensibilidad. Trabajos previous describen una alta correlación entre las técnicas de SN vs IFI y de SN vs ELISA, destacando una mayor sensibilidad para el ELISA. El presente trabajo compara la sensibilidad y especificidad de IFI, ELISA y SN en la detección de anticuerpos a BHV-1 utilizando 105 sueros bovinos. La especificidad de las técnicas comparadas se demostró por la seroconversión obtenida en sueros de animales experimentalmente infectados (Cuadro 1). Se observó una alta asociación entre sueros reactivos y no reactivos entre SN e IFI, SN y ELISA y entre Ifi y ELISA destacándose una mayor sensibilidad para el ELISA (Figuras 2 y 3). El estudio estadístico de los resultados obtenidos con las tres técnicas diagnósticas determinó un alto coeficiente de correlación entre las mismas (Cuadro 2). Debido a la simplicidad y facilidad de ejecución se recomienda la técnica de ELISA para estudios epizootiológicos de larga escala

Comparison of three serological techniques for the diagnosis of bovine herpesvirus type 1: serum neutralization, enzyme-linked immunosorbent assay and indirect immunofluorescence.

Serum neutralization (SN), indirect immunofluorescence (IFI) and enzyme-linked immunosorbent assay (ELISA) were compared for their sensitivity and specificity to detect bovine serum antibodies to Bovine Herpesvirus type 1 on 105 bovine serum samples. High correlation coefficients among all three techniques were observed, showing a higher sensitivity for the ELISA test. Due to its simplicity and its feasibility to be adopted in less equipped laboratories, the ELISA test is recommended for large scale epizotiological studies, serological diagnosis and detection of reactive animals.

Analysis of the transcriptional promoter which regulates the latency-related transcript of bovine herpesvirus 1.

As a transcriptional promoter in primary cultures of sensory ganglionic neurons, DNA sequences near the 5' terminus of the latency-related (LR) gene of bovine herpesvirus 1 were at least 10 times more efficient than the simian virus 40 early promoter-enhancer. In contrast, as a promoter in bovine, rodent, or monkey cells, the LR promoter was approximately six times less efficient than the simian virus 40 early promoter-enhancer. The LR promoter had strict orientation preferences in neurons and all other mammalian cell lines tested. Removal of a 146-base-pair XhoI fragment from the LR promoter resulted in stimulation of LR promoter activity in bovine cells but not rabbit neurons, monkey fibroblasts, or rodent cells. LR promoter activity in bovine cells is stimulated by bovine herpesvirus 1 lytic infection, suggesting that viral gene products or virus-induced factors positively regulate the expression of the LR gene. A synthetic glucocorticoid, dexamethasone, repressed LR promoter activity in bovine cells. These results imply that a variety of factors can influence the expression of the LR gene during latent infections of neurons as well as during the lytic infection cycle.

Comparison of three serological techniques for the diagnosis of bovine herpesvirus type 1: serum neutralization, enzyme-linked immunosorbent assay and indirect immunofluorescence.

Serum neutralization (SN), indirect immunofluorescence (IFI) and enzyme-linked immunosorbent assay (ELISA) were compared for their sensitivity and specificity to detect bovine serum antibodies to Bovine Herpesvirus type 1 on 105 bovine serum samples. High correlation coefficients among all three techniques were observed, showing a higher sensitivity for the ELISA test. Due to its simplicity and its feasibility to be adopted in less equipped laboratories, the ELISA test is recommended for large scale epizotiological studies, serological diagnosis and detection of reactive animals.