Genomic deletions and mutations resulting in the loss of eight genes reduce the in vivo replication capacity of Meleagrid herpesvirus 1.

Meleagrid herpesvirus 1 (MeHV-1 or turkey herpesvirus) has been widely used as a vaccine in commercial poultry. Initially, these vaccine applications were for the prevention of Marek's disease resulting from Gallid herpesvirus 2 infections, while more recently MeHV-1 has been used as recombinant vector for other poultry infections. The construction of herpesvirus infectious clones that permit propagation and manipulation of the viral genome in bacterial hosts has advanced the studies of herpesviral genetics. The current study reports the construction of five MeHV-1 infectious clones. The in vitro properties of viruses recovered from these clones were indistinguishable from the parental MeHV-1. In contrast, the rescued MeHV-1 viruses were significantly attenuated when used in vivo. Complete sequencing of the infectious clones identified the absence of two regions of the MeHV-1 genome compared to the MeHV-1 reference sequence. These analyses determined the rescued viruses have seven genes, UL43, UL44, UL45, UL56, HVT071, sorf3 and US2 either partially or completely deleted. In addition, single nucleotide polymorphisms were identified in all clones compared with the MeHV-1 reference sequence. As a consequence of one of the polymorphisms identified in the UL13 gene, four of the rescued viruses were predicted to encode a serine/threonine protein kinase lacking two of three domains required for activity. Thus four of the recovered viruses have a total of eight missing or defective genes. The implications of these findings in the context of herpesvirus biology and infectious clone construction are discussed.

Characterisation of the Upper Respiratory Tract Virome of Feedlot Cattle and Its Association with Bovine Respiratory Disease.

Bovine respiratory disease (BRD) is a major health problem within the global cattle industry. This disease has a complex aetiology, with viruses playing an integral role. In this study, metagenomics was used to sequence viral nucleic acids in the nasal swabs of BRD-affected cattle. The viruses detected included those that are well known for their association with BRD in Australia (bovine viral diarrhoea virus 1), as well as viruses known to be present but not fully characterised (bovine coronavirus) and viruses that have not been reported in BRD-affected cattle in Australia (bovine rhinitis, bovine influenza D, and bovine nidovirus). The nasal swabs from a case-control study were subsequently tested for 10 viruses, and the presence of at least one virus was found to be significantly associated with BRD. Some of the more recently detected viruses had inconsistent associations with BRD. Full genome sequences for bovine coronavirus, a virus increasingly associated with BRD, and bovine nidovirus were completed. Both viruses belong to the Coronaviridae family, which are frequently associated with disease in mammals. This study has provided greater insights into the viral pathogens associated with BRD and highlighted the need for further studies to more precisely elucidate the roles viruses play in BRD.

In Vivo Characterisation of Five Strains of Bovine Viral Diarrhoea Virus 1 (Subgenotype 1c).

Bovine viral diarrhoea virus 1 (BVDV-1) is strongly associated with several important diseases of cattle, such as bovine respiratory disease, diarrhoea and haemoragic lesions. To date many subgenotypes have been reported for BVDV-1, currently ranging from subgenotype 1a to subgenotype 1u. While BVDV-1 has a world-wide distribution, the subgenotypes have a more restricted geographical distribution. As an example, BVDV-1 subgenotypes 1a and 1b are frequently detected in North America and Europe, while the subgenotype 1c is rarely detected. In contrast, BVDV-1 subgenotype 1c is by far the most commonly reported in Australia. Despite this, uneven distribution of the biological importance of the subgenotypes remains unclear. The aim of this study was to characterise the in vivo properties of five strains of BVDV-1 subgenotype 1c in cattle infection studies. No overt respiratory signs were reported in any of the infected cattle regardless of strain. Consistent with other subgenotypes, transient pyrexia and leukopenia were commonly identified, while thrombocytopenia was not. The quantity of virus detected in the nasal secretions of transiently infected animals suggested the likelihood of horizontal transmission was very low. Further studies are required to fully understand the variability and importance of the BVDV-1 subgenotype 1c.

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.

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.

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.

The essential and non-essential genes of Bovine herpesvirus 1.

Bovine herpesvirus 1 (BoHV-1) is an economically important pathogen of cattle associated with respiratory and reproductive disease. To further develop BoHV-1 as a vaccine vector, a study was conducted to identify the essential and non-essential genes required for in vitro viability. Random-insertion mutagenesis utilizing a Tn5 transposition system and targeted gene deletion were employed to construct gene disruption and gene deletion libraries, respectively, of an infectious clone of BoHV-1. Transposon insertion position and confirmation of gene deletion were determined by direct sequencing. The essential or non-essential requirement of either transposed or deleted open reading frames (ORFs) was assessed by transfection of respective BoHV-1 DNA into host cells. Of the 73 recognized ORFs encoded by the BoHV-1 genome, 33 were determined to be essential and 36 to be non-essential for virus viability in cell culture; determining the requirement of the two dual copy ORFs was inconclusive. The majority of ORFs were shown to conform to the in vitro requirements of BoHV-1 homologues encoded by human herpesvirus 1 (HHV-1). However, ORFs encoding glycoprotein K (UL53), regulatory, membrane, tegument and capsid proteins (UL54, UL49.5, UL49, UL35, UL20, UL16 and UL7) were shown to differ in requirement when compared to HHV-1-encoded homologues.

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.