title: Development of a real-time reverse transcription PCR assay for detection of a novel nidovirus associated with a neurological disease of the Australian brushtail possum (Trichosurus vulpecula).
abstract: OBJECTIVE
  To develop a quantitative reverse transcription PCR (RT-qPCR) assay for detection of the putative wobbly possum disease (WPD) virus and to apply this test to investigate the viral load in archival tissues from past WPD transmission studies.
  
  
  METHODS
  The real-time assay was developed as a two-step RT-qPCR in a SYBR green format and validated using serial dilutions of a linearised plasmid containing target DNA. The copy number values were normalised to the amount of RNA in each reverse transcription reaction and presented as the number of viral copies per μg of total [corrected] RNA. The viral load was determined in archival samples from animals that had received inoculations of infectious WPD tissue suspensions. Thirty samples originating from 22 possums, comprising five samples from three healthy possums and 25 samples from 19 possums that had received inoculations of infectious WPD tissue suspensions were tested.
  
  
  RESULTS
  The assay was linear (R(2) > 0.99) within the tested range from 1 to 10(7) target copies/µL, with an efficiency of >90%. The intra-assay variability CV values ranged from 0.8 to 4.5% for different standards, with the inter-assay variability CV values ranging from 0.4 to 2.5%, indicating good precision and reproducibility of the assay. The novel nidovirus was detected in all 25 samples from WPD-affected possums. Tissues from three control possums and from one experimentally infected rabbit were negative for WPD RNA. The viral load in WPD-positive tissues differed between individual possums and between tissue types, ranging from 2.2 to 359,980 copies/pg RNA. The highest viral load was detected in liver, followed by brain, spleen, kidney and urine. There was a more than four log difference in the viral load between pools of tissues originating from two outbreaks of WPD in different geographical regions.
  
  
  CONCLUSIONS
  Detection of viral RNA in a variety of tissues from WPD affected possums, including brain, is consistent with the multi-organ distribution of histopathological lesions observed in WPD. Our data suggest that liver may constitute the sample of choice for diagnostic testing. Differences in the viral load in tissues from possums inoculated with infectious WPD tissue suspensions from Rotorua or Invermay origin suggest that WPD viruses with different biological properties may exist.
  
  
  CONCLUSIONS
  We have developed a RT-qPCR assay for detection of the putative WPD virus. The test showed good sensitivity and reproducibility over the wide dynamic range of template concentrations. It provides a tool for future diagnostic and research purposes.
authors.affiliation: Institute of Veterinary Animal and Biomedical Sciences, Massey University, Palmerston North, New Zealand. M.Dunowska@massey.ac.nz
authors.forename: M
authors.initials: M
authors.lastname: Dunowska
authors.forename: G
authors.initials: G
authors.lastname: Gopakumar
authors.forename: M R
authors.initials: MR
authors.lastname: Perrott
mesh_terms: D000818:Animals; D001921:Brain / Q000821:virology; D002493:Central Nervous System Diseases / Q000175:diagnosis / Q000662:veterinary* / Q000821:virology; D007668:Kidney / Q000821:virology; D008099:Liver / Q000821:virology; D028381:Nidovirales / Q000302:isolation & purification*; D030341:Nidovirales Infections / Q000175:diagnosis / Q000662:veterinary* / Q000821:virology; D060888:Real-Time Polymerase Chain Reaction / Q000379:methods / Q000662:veterinary*; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods / Q000662:veterinary*; D013154:Spleen / Q000821:virology; D048330:Trichosurus; D014556:Urine / Q000821:virology