Aneta Gubala

Molecular-Beacon Multiplex Real-Time PCR Assay for Detection of Vibrio cholerae

ABSTRACT A multiplex real-time PCR assay was developed using molecular beacons for the detection of Vibrio cholerae by targeting four important virulence and regulatory genes. The specificity and sensitivity of this assay, when tested with pure culture and spiked environmental water samples, were high, surpassing those of currently published PCR assays for the detection of this organism.

Genomic characterisation of Wongabel virus reveals novel genes within the Rhabdoviridae

Viruses belonging to the family Rhabdoviridae infect a variety of different hosts, including insects, vertebrates and plants. Currently, there are approximately 200 ICTV-recognised rhabdoviruses isolated around the world. However, the majority remain poorly characterised and only a fraction have been definitively assigned to genera. The genomic and transcriptional complexity displayed by several of the characterised rhabdoviruses indicates large diversity and complexity within this family. To enable an improved taxonomic understanding of this family, it is necessary to gain further information about the poorly characterised members of this family. Here we present the complete genome sequence and predicted transcription strategy of Wongabel virus (WONV), a previously uncharacterised rhabdovirus isolated from biting midges (Culicoides austropalpalis) collected in northern Queensland, Australia. The 13,196 nucleotide genome of WONV encodes five typical rhabdovirus genes N, P, M, G and L. In addition, the WONV genome contains three genes located between the P and M genes (U1, U2, U3) and two open reading frames overlapping with the N and G genes (U4, U5). These five additional genes and their putative protein products appear to be novel, and their functions are unknown. Predictive analysis of the U5 gene product revealed characteristics typical of viroporins, and indicated structural similarities with the alpha-1 protein (putative viroporin) of viruses in the genus Ephemerovirus. Phylogenetic analyses of the N and G proteins of WONV indicated closest similarity with the avian-associated Flanders virus; however, the genomes of these two viruses are significantly diverged. WONV displays a novel and unique genome structure that has not previously been described for any animal rhabdovirus.

Genetic and epidemiological characterization of Stretch Lagoon orbivirus, a novel orbivirus isolated from Culex and Aedes mosquitoes in northern Australia

Stretch Lagoon orbivirus (SLOV) was isolated in 2002 from pooled Culex annulirostris mosquitoes collected at Stretch Lagoon, near the Wolfe Creek national park in the Kimberley region of Western Australia. Conventional serological tests were unable to identify the isolate, and electron microscopy indicated a virus of the genus Orbivirus, family Reoviridae. Here, a cDNA subtraction method was used to obtain approximately one-third of the viral genome, and further sequencing was performed to complete the sequences of segment 1 (viral polymerase) and segment 2 (conserved inner-core protein). Phylogenetic analysis showed that SLOV should be considered a new species within the genus Orbivirus. A real-time RT-PCR test was designed to study the epidemiology of SLOV in the field. Six additional isolates of SLOV were identified, including isolates from four additional locations and two additional mosquito species. Horses, donkeys and goats were implicated as potential vertebrate hosts in a serological survey.

Genomic Characterisation of Three Mapputta Group Viruses, a Serogroup of Australian and Papua New Guinean Bunyaviruses Associated with Human Disease

The Mapputta serogroup tentatively contains the mosquito-associated viruses Mapputta, Maprik, Trubanaman and Gan Gan. Interestingly, this serogroup has previously been associated with an acute epidemic polyarthritis-like illness in humans; however, there has been no ensuing genetic characterisation. Here we report the complete genome sequences of Mapputta and Maprik viruses, and a new Mapputta group candidate, Buffalo Creek virus, previously isolated from mosquitoes and detected by serology in a hospitalised patient. Phylogenetic analyses indicate that the group is one of the earliest diverged groups within the genus Orthobunyavirus of the family Bunyaviridae. Analyses show that these three viruses are related to the recently sequenced Australian bunyaviruses from mosquitoes, Salt Ash and Murrumbidgee. A notable feature of the Mapputta group viruses is the absence of the NSs (non-structural) ORF commonly found on the S segment of other orthobunyaviruses. Viruses of the Mapputta group have been isolated from geographically diverse regions ranging from tropical Papua New Guinea to the semi-arid climate of south-eastern Australia. The relevance of this group to human health in the region merits further investigation.

Persistent and recrudescent infection in cattle following natural infection with Middle Point orbivirus

Middle Point orbivirus (MPOV) is a recently described Australian arbovirus, related to Yunnan orbivirus from China. Analysis of genetic variation within the major serotype gene of MPOV isolates collected from sentinel cattle has identified eight co-circulating strains. The pattern of strain isolation from individual animals during the study period was consistent with an interpretation of persistent MPOV infection of up to five months, featuring episodes of quiescence (below levels required for virus isolation) followed by viral recrudescence. This is significant with regard to current interpretations of infection, persistence and recrudescence during natural infections of orbiviruses, including bluetongue virus.

Genomic Characterisation of Vinegar Hill Virus, An Australian Nairovirus Isolated in 1983 from Argas Robertsi Ticks Collected from Cattle Egrets

This report describes the near complete genomic sequence and subsequent analysis of Vinegar Hill virus (VINHV; tentative member of the genus Orthonairovirus, family Nairoviridae, order Bunyavirales). VINHV is the second nairovirus reported to be isolated on mainland Australia and the first to be sequenced and analysed. Our genetic analysis shows that VINHV belongs to the Dera Ghazi Khan genogroup, a group of viruses previously isolated in other parts of the world including Asia, South Africa, and the USA. We discuss possible routes of entry for nairoviruses into Australia and the need to understand the virome of Australian ticks in the context of new and emerging disease.

Identification of very small open reading frames in the genomes of Holmes Jungle virus, Ord River virus, and Wongabel virus of the genus Hapavirus, family Rhabdoviridae:

Viruses of the family Rhabdoviridae infect a broad range of hosts from a variety of ecological and geographical niches, including vertebrates, arthropods, and plants. The arthropod-transmitted members of this family display considerable genetic diversity and remarkable genomic flexibility that enable coding for various accessory proteins in different locations of the genome. Here, we describe the genome of Holmes Jungle virus, isolated from Culex annulirostris mosquitoes collected in northern Australia, and make detailed comparisons with the closely related Ord River and Wongabel viruses, with a focus on identifying very small open reading frames (smORFs) in their genomes. This is the first systematic prediction of smORFs in rhabdoviruses, emphasising the intricacy of the rhabdovirus genome and the knowledge gaps. We speculate that these smORFs may be of importance to the life cycle of the virus in the arthropod vector.

Identification of very small open reading frames in the genomes of Holmes Jungle virus, Ord River virus, and Wongabel virus of the genus , family

Viruses of the family Rhabdoviridae infect a broad range of hosts from a variety of ecological and geographical niches, including vertebrates, arthropods, and plants. The arthropod-transmitted members of this family display considerable genetic diversity and remarkable genomic flexibility that enable coding for various accessory proteins in different locations of the genome. Here, we describe the genome of Holmes Jungle virus, isolated from Culex annulirostris mosquitoes collected in northern Australia, and make detailed comparisons with the closely related Ord River and Wongabel viruses, with a focus on identifying very small open reading frames (smORFs) in their genomes. This is the first systematic prediction of smORFs in rhabdoviruses, emphasising the intricacy of the rhabdovirus genome and the knowledge gaps. We speculate that these smORFs may be of importance to the life cycle of the virus in the arthropod vector.