Expression of tlr4, md2 and cd14 in equine blood leukocytes during endotoxin infusion and in intestinal tissues from healthy horses.

The expression of tlr4, md2 and cd14 was studied in equine blood leukocytes and in intestinal samples using real time PCR. The stability of three commonly used reference genes, glyceraldehyde-3P-dehydrogenase (GAPDH), hypoxantine ribosyltransferase (HPRT) and succinate dehydrogenase complex subunit A (SDHA), was evaluated using qbase(PLUS). The equine peripheral blood mononuclear cells (eqPBMC) examined were either stimulated in vitro with Phorbol 12-myristate 13-acetate (PMA) and ionomycin or with the CpG oligodeoxynuclotide 2216 (CpG-ODN 2216) or obtained from horses before, during and after infusion of endotoxin. Intestinal tissue from healthy horses was sampled at ileum, right dorsal colon and rectum. Ranking of the three reference genes used for normalisation identified the combination HPRT/SDHA as most suitable both when determined ex vivo in leukocytes obtained from experimentally induced endotoxaemia and in eqPBMC activated in vitro while HPRT/GAPDH were most appropriate for the intestinal samples. The relative amounts of mRNA for TLR4 and MD-2 increased threefold during in vitro activation of the cells with CpG-ODN 2216 but was decreased in cultures stimulated with PMA/ionomycin. A transient elevation in the transcription of tlr4 and md2 was also evident for equine blood leukocytes following endotoxaemia. The levels of mRNA for CD14 on the other hand remained unaffected both during the induction of endotoxaemia and in the in vitro stimulated PBMCs. A low steady expression of TLR4, MD-2 and CD14 mRNA was demonstrated for the intestinal samples with no variation between the intestinal segments analysed. Thus, the foundation for real time PCR based levels of analysis of mRNA for all three components in the equine LPS receptor complex in different intestinal segments was set, making it possible to carry out future expression studies on clinical material.

Development and comparison of a Primer-Probe Energy Transfer based assay and a 5' conjugated Minor Groove Binder assay for sensitive real-time PCR detection of infectious laryngotracheitis virus.

In this study the design and development of two real-time PCR assays for the rapid, sensitive and specific detection of infectious laryngotracheitis virus (ILTV) DNA is described. A Primer-Probe Energy Transfer (PriProET) assay and 5' conjugated Minor Groove Binder (MGB) method are compared and contrasted. Both have been designed to target the thymidine kinase gene of the ILTV genome. Both PriProET and MGB assays are capable of detecting 20 copies of a DNA standard per reaction and are linear from 2×10(8) to 2×10(2)copies/µl. Neither PriProET, nor MGB reacted with heterologous herpesviruses, indicating a high specificity of the two methods as novel tools for virus detection and identification. This study demonstrates the suitability of PriProET and 5' conjugated MGB probes as real-time PCR chemistries for the diagnosis of respiratory diseases caused by ILTV.

Development of a minor groove binder assay for real-time one-step RT-PCR detection of swine vesicular disease virus.

The design and development of a 5' conjugated minor groove binder (MGB) probe real-time RT-PCR assay are described for rapid, sensitive and specific detection of swine vesicular disease virus (SVDV) RNA. The assay is designed to target the 2C gene of the SVDV genome and is capable of detecting 2×10(2) copies of an RNA standard per reaction. It does not detect any of the other RNA viruses that cause vesicular disease in pigs, or the human enterovirus, Coxsackie B5 virus (CVB5) which is closely related antigenically to SVDV. The linear range of this test was from 2×10(2) to 2×10(8) copies/µl. The assay is rapid and can detect SVDV RNA in just over 3.5 h including the time required for nucleic acid extraction. The development of this assay provides a useful tool for the differential diagnosis of SVD or for the detection of SVDV in research applications. This study demonstrates the suitability of MGB probes as a real-time PCR chemistry for the diagnosis of swine vesicular disease.

Interaction of classical swine fever virus with dendritic cells.

Functional disruption of dendritic cells (DCs) is an important strategy for viral pathogens to evade host defences. Monocytotropic viruses such as classical swine fever virus (CSFV) could employ such a mechanism, since the virus can suppress immune responses and induce apoptosis without infecting lymphocytes. Here, CSFV was shown to infect and efficiently replicate in monocyte- and in bone marrow-derived DCs. Interestingly, the infected DCs displayed neither modulated MHC nor CD80/86 expression. Stimulation of DCs with IFN-alpha/TNF-alpha or polyinosinic-polycytidylic acid (pIC) induced phenotypic maturation with increased MHC and CD80/86 expression, both with mock-treated and infected DCs. In addition, the T cell stimulatory capacity of CSFV-infected DCs was maintained both in a polyclonal T cell stimulation and in specific antigen-presentation assays, requiring antigen uptake and processing. Interestingly, similar to macrophages, CSFV did not induce IFN-alpha responses in these DCs and even suppressed pIC-induced IFN-alpha induction. Other cytokines including interleukin (IL)-6, IL-10, IL-12 and TNF-alpha were not modulated. Taken together, these results demonstrated that CSFV can replicate in DCs and control IFN type I responses, without interfering with the immune reactivity. These results are interesting considering that DC infection with RNA viruses usually results in DC activation.

Porcine rubulavirus LPMV RNA persists in the central nervous system of pigs after recovery from acute infection.

In order to study persistence of the porcine rubulavirus LPMV, we examined tissue samples collected from pigs 53 days after experimental infection. These pigs survived the initial infection and could clinically be considered to have recovered from the infection. Two of the pigs used in this study were chemically immunosuppressed during the last 4 days before necropsy. No infectious virus or viral antigen could be detected in any tissue using standard methods for virus isolation and detection. However, the presence of viral genomic RNA and mRNA could be demonstrated in the mid brain of the convalescent pig using an optimised RT-nested PCR. Mid brain, forebrain and lung were all shown to contain LPMV RNA in the immunosuppressed convalescent pigs. In addition we examined the P-gene editing in the recovered pigs and conclude that the viral genome is transcriptionally active in these pigs. The relevance of the persistence of LPMV for maintenance and spread within and/or between pig populations is discussed.

Multiple factors including subgenomic RNAs and reduced viral protein expression are associated with a persistent infection by porcine rubulavirus (LPMV).

The synthesis of virus specific RNA and the expression of viral proteins in PK-15 cells persistently infected with the porcine rubulavirus LPMV have been studied at two different cell-passages following establishment of persistency (passages 25 and 65). Protein analysis of persistently infected cells and the virus particles released from these failed to demonstrate the presence of the polymerase (L) protein. A decrease in the amount of the phospho- (P) protein was also noted. The genome and mRNAs, both mono- and bicistronic, could readily be identified in the persistently infected cells with the exception of the L mRNA. By analysis of transcription gradients generated using the NIH Image analysis software, as well as analysis of the editing frequency, it was concluded that the changes in viral protein levels in persistently infected cells could be associated with a reduction in the amount of L mRNA and a shift in editing of the P gene. In addition, several large subgenomic RNAs of both the internally deleted and copy-back type were found in the persistently infected cells. The relevance of these findings to the persistent state is discussed.

Establishment and characterisation of a porcine rubulavirus (LPMV) persistent infection in porcine kidney cells.

Porcine rubulavirus (LPMV) can establish persistent infections in porcine kidney cells. Cell cultures characterised at passages 25 and 65 demonstrated haemadsorption, formation of syncytia, and a slower growth rate. The nucleoprotein (NP) and haemagglutinin-neuraminidase (HN) protein were present in all cells, although not to the same extent as in wild type infected cells. Incubation of the cell cultures with virus neutralising antibodies could not cure them from the infection. The cells were resistant to LPMV high multiplicity superinfection, but lysed rapidly upon infection with VSV. These cells thus fulfilled the criteria of a true persistent infection. Viral particles were released into the medium from the persistently infected cells as measured by HA and infection of PK-15 cells with medium from the persistently infected cells. The infectious titer of the virus released from the persistently infected cells was 3 logs lower compared to wild type virus, the HN titer still being comparable. Virus released from the persistently infected cells was unable to cause a lytic infection in PK-15 cells, and showed a reduced ability to spread when compared to a LPMV lytic infection.

The molecular biology of the porcine paramyxovirus LPMV.

Protein and genomic studies of a previously uncharacterized porcine paramyxovirus (designated LPMV) confirmed that it was a member of the paramyxovirus genus. The nucleotide sequences and deduced amino acids of the complete P-gene, M-gene, F-gene and HN-gene as well as the intergenic sequences have been determined. Comparative sequence analysis of the M-gene of LPMV revealed the closest relationship of LPMV was to human mumps virus with a homology of 46% and 55% at the amino acid and nucleotide levels respectively. The P-gene of LPMV is transcribed to V protein mRNA and by editing of the gene to the P protein mRNA. The LPMV P-gene has the coding capacity for an additional protein of 126 amino acids, a C protein.

The P gene of the porcine paramyxovirus LPMV encodes three possible polypeptides P, V and C: the P protein mRNA is edited.

The nucleotide sequence of the P gene of the porcine paramyxovirus La-Piedad-Michoacan-Mexico virus (LPMV) was analysed. Three long open reading frames (ORFs) were found in the mRNA sense. Insertion of two G residues is necessary to obtain an ORF encoding the P protein, which gives a P protein of 404 amino acids with a calculated Mr of 42475. This form of editing was demonstrated, two non-templated G residues being added in a portion of the mRNA transcripts. The LPMV V protein, which has a conserved cysteine-rich C-terminal region, is encoded by an exact copy of the P gene. The third ORF has the capacity to encode a protein of 126 amino acids, which may resemble the C proteins found in some paramyxoviruses. The ORF starts from an AUG codon down-stream of the first AUG codon of the P/V ORF.