The BMP2 nuclear variant, nBMP2, is expressed in mouse hippocampus and impacts memory.

The novel nuclear protein nBMP2 is synthesized from the BMP2 gene by translational initiation at an alternative start codon. We generated a targeted mutant mouse, nBmp2NLStm, in which the nuclear localization signal (NLS) was inactivated to prevent nuclear translocation of nBMP2 while still allowing the normal synthesis and secretion of the BMP2 growth factor. These mice exhibit abnormal muscle function due to defective Ca2+ transport in skeletal muscle. We hypothesized that neurological function, which also depends on intracellular Ca2+ transport, could be affected by the loss of nBMP2. Age-matched nBmp2NLStm and wild type mice were analyzed by immunohistochemistry, behavioral tests, and electrophysiology to assess nBMP2 expression and neurological function. Immunohistochemical staining of the hippocampus detected nBMP2 in the nuclei of CA1 neurons in wild type but not mutant mice, consistent with nBMP2 playing a role in the hippocampus. Mutant mice showed deficits in the novel object recognition task, suggesting hippocampal dysfunction. Electrophysiology experiments showed that long-term potentiation (LTP) in the hippocampus, which is dependent on intracellular Ca2+ transport and is thought to be the cellular equivalent of learning and memory, was impaired. Together, these results suggest that nBMP2 in the hippocampus impacts memory formation.

Absolute quantitation of Marek's disease virus and Herpesvirus of turkeys in chicken lymphocyte, feather tip and dust samples using real-time PCR.

The further development of Taqman quantitative real-time PCR (qPCR) assays for the absolute quantitation of Marek's disease virus serotype 1 (MDV1) and Herpesvirus of turkeys (HVT) viruses is described and the sensitivity and reproducibility of each assay reported. Using plasmid DNA copies, the lower limit of detection was determined to be 5 copies for the MDV1 assay and 75 copies for the HVT assay. Both assays were found to be highly reproducible for Ct values and calculated copy numbers with mean intra- and inter-assay coefficients of variation being less than 5% for Ct and 20% for calculated copy number. The genome copy number of MDV1 and HVT viruses was quantified in PBL and feather tips from experimentally infected chickens, and field poultry dust samples. Parallelism was demonstrated between the plasmid-based standard curves, and standard curves derived from infected spleen material containing both viral and host DNA, allowing the latter to be used for absolute quantification. These methods should prove useful for the reliable differentiation and absolute quantitation of MDV1 and HVT viruses in a wide range of samples.

Genetic analysis of bovine viral diarrhoea viruses from Australia.

Eighty-nine bovine viral diarrhoea viruses (BVDV) from Australia have been genetically typed by sequencing of the 5' untranslated region (5'-UTR) and for selected isolates the N(pro) region of the viral genome. Phylogenetic reconstructions indicated that all of the samples examined clustered within the BVDV type 1 genotype. Of the 11 previously described genetic groups of BVDV-1, 87 of the samples examined in this study clustered with the BVDV-1c, while two samples clustered with the BVDV-1a. Based on these analyses there appears to be limited genetic variation within the Australian BVDV field isolates. In addition, the phylogenetic reconstructions indicate that the clustering of Australian BVDV in the phylogenetic trees is not a result of geographic isolation.

Construction of a gene inactivation library for Bovine herpesvirus 1 using infectious clone technology.

The application of infectious clone technology to herpesvirus biology has revolutionized the study of these viruses. Previously the ability to manipulate these large DNA viruses was limited to methods dependent on homologous recombination in mammalian cells. However, the construction of herpesvirus infectious clones using bacterial artificial chromosome vectors has permitted the application of powerful bacterial genetics for the manipulation of these viruses. A method is described for the construction and characterization of a gene inactivation library of Bovine herpesvirus 1 using an infectious clone. The method utilizes transposon-mediated gene inactivation, which permits gene inactivation without any prior knowledge of the viral genomic sequence. Furthermore, as the genetic manipulation is performed in bacteria the inactivation of those viral genes that are essential for viral replication is also possible. The method described here can be readily applied to any herpesvirus clone and provides the tools for precise characterization of all the genes contained within a herpesvirus genome.

Differential amplification and quantitation of Marek's disease viruses using real-time polymerase chain reaction.

Quantitative real-time PCR (qPCR) assays for the three serotypes of Marek's disease virus (MDV) have been developed. An internal control qPCR assay that detects chicken alpha2 (VI) collagen gene was also developed to allow quantitation of MDV. To reduce costs and time, the assays for MDV1 and the internal control were combined into a duplex assay. The sensitivity, specificity, precision, and reproducibility of each assay are reported. The MDV qPCR assays were specific to their target gene when compared using Australian field and vaccine strains of MDV and 10-100-fold more sensitive than standard PCR. Using DNA from infected spleen tissue, the lower limit of detection of total DNA (viral and host combined) was 0.025 ng for the MDV1 and collagen assays, and 0.25 ng for the HVT and MDV2 assays. All assays were found to be highly reproducible for Ct values, but less so for calculated concentrations. MDV1 and HVT were quantitated in spleen tissue of twenty experimentally infected chickens 7-35 days after infection. The relative abundance of MDV1 exhibited a clear peak at day 14 post-infection, whereas HVT displayed an increasing trend over the 35 days post-infection. The duplex assay was optimized such that it was able to accurately quantitate MDV1 in samples of very high, medium, and very low relative abundance of MDV1. These qPCR assays will be useful for reliable differentiation and quantitation of MDV for a range of research and industry applications.

A molecular epidemiological study of Australian bat lyssavirus.

The genetic diversity of Australian bat lyssavirus (ABL) was investigated by comparing 24 ABL isolate glycoprotein (G) gene nucleotide sequences with those of 37 lyssaviruses representing Lyssavirus genotypes 1-6. Phylogenetic analyses indicated that ABL forms a monophyletic group separate from other lyssaviruses. This group differentiates into two clades: one associated with Pteropus (flying fox) species, the other with the insectivorous bat Saccolaimus flaviventris. Calculation of percentage nucleotide identities between isolates of the two clades revealed up to 18.7 % nucleotide sequence divergence between the two ABL variants. These observations suggest that ABL is a separate lyssavirus species with a similar epidemiology to chiropteran rabies virus (RV), where two distinct ABL variants co-exist in Australia in bat species with dissimilar ecology. Analyses of selection pressures in ABL G gene sequences provided some evidence of weak positive selection within the endodomain at amino acids 499 and 501, although in general the dominant evolutionary process observed was purifying selection. This intimates that, in nature, isolates of ABL, like those of RV, are subject to relatively strong selective constraints, suggesting a stability of host species, cell tropisms and ecological conditions.

Rapid and efficient construction of recombinant bovine herpesvirus 1 genomes.

Bovine herpesvirus 1 (BoHV-1) is an important pathogen of cattle. Recombinant bovine herpesvirus 1 viruses (rBoHV) have been studied extensively as potential vaccines for BoHV-1 associated diseases. A method is described which advances protocols used currently for constructing rBoHV by producing recombinant viruses free of parent virus. The method, restriction endonuclease mediated recombination (REMR), utilises a unique NsiI site in the BoHV-1 genome. Following NsiI digestion the two genomic fragments are prevented from recombining by dephosphorylation. However, when the genomic fragments are co-transfected into a susceptible cell-line with a third DNA fragment (DNA bridge), which encodes DNA homologous to the digested viral termini, the three DNA molecules are able to undergo homologous recombination and produce infectious BoHV-1. During the recombination process foreign DNA within the DNA bridge is incorporated into the BoHV-1 genome, producing rBoHV. In the absence of the DNA bridge virus reconstitution does not occur thus eliminating contamination by the nonrecombinant parent virus. As REMR used an NsiI site occurring naturally in the BoHV-1 genome it can be used for the insertion of foreign DNA into the genome without any prior modifications. REMR could also be applied to any herpesvirus for which the genome sequence is known.

Construction and manipulation of an infectious clone of the bovine herpesvirus 1 genome maintained as a bacterial artificial chromosome.

The complete genome of bovine herpesvirus 1 (BoHV-1) strain V155 has been cloned as a bacterial artificial chromosome (BAC). Following electroporation into Escherichia coli strain DH10B, the BoHV-1 BAC was stably propagated over multiple generations of its host. BAC DNA recovered from DH10B cells and transfected into bovine cells produced a cytopathic effect which was indistinguishable from that of the parent virus. Analysis of the replication kinetics of the viral progeny indicated that insertion of the BAC vector into the thymidine kinase gene did not affect viral replication. Specific manipulation of the BAC was demonstrated by deleting the gene encoding glycoprotein E by homologous recombination in DH10B cells facilitated by GET recombination. These studies illustrate that the propagation and manipulation of herpesviruses in bacterial systems will allow for rapid and accurate characterization of BoHV-1 genes. In turn, this will allow for the full utilization of BoHV-1 as a vaccine vector.