title: Development of real-time reverse transcriptase qPCR assays for the detection of Punta Toro virus and Pichinde virus.
abstract: BACKGROUND
  Research with high biocontainment pathogens such as Rift Valley fever virus (RVFV) and Lassa virus (LASV) is expensive, potentially hazardous, and limited to select institutions. Surrogate pathogens such as Punta Toro virus (PTV) for RVFV infection and Pichinde virus (PICV) for LASV infection allow research to be performed under more permissive BSL-2 conditions. Although used as infection models, PTV and PICV have no standard real-time RT-qPCR assays to detect and quantify pathogenesis. PTV is also a human pathogen, making a standardized detection assay essential for biosurveillance. Here, we developed and characterized two real-time RT-qPCR assays for PICV and PTV by optimizing assay conditions and measuring the limit of detection (LOD) and performance in multiple clinical matrices.
  
  
  METHODS
  Total nucleic acid from virus-infected Vero E6 cells was used to optimize TaqMan-minor groove binder (MGB) real-time RT-qPCR assays. A 10-fold dilution series of nucleic acid was used to perform analytical experiments with 60 replicates used to confirm assay LODs. Serum and whole blood spiked with 10-fold dilutions of PTV and PICV virus were assessed as matrices in a mock clinical context. The Cq, or cycle at which the fluoresce of each sample first crosses a threshold line, was determined using the second derivative method using Roche LightCycler 480 software version 1.5.1. Digital droplet PCR (ddPCR) was utilized to quantitatively determine RNA target counts/μl for PTV and PICV.
  
  
  RESULTS
  Optimized PTV and PICV assays had LODs of 1000 PFU/ml and 100 PFU/ml, respectively, and this LOD was confirmed in 60/60 (PTV) and 58/60 (PICV) positive replicates. Preliminary mock clinical LODs remained consistent in serum and whole blood for PTV and PICV at 1000 PFU/ml and 100 PFU/ml. An exclusivity panel showed no cross reaction with near neighbors.
  
  
  CONCLUSIONS
  PTV and PICV Taq-man MGB based real-time RT-qPCR assays developed here showed relevant sensitivity and reproducibility in samples extracted from a variety of clinical matrices. These assays will be useful as a standard by researchers for future experiments utilizing PTV and PICV as infection models, offering the ability to track infection and viral replication kinetics during research studies.
authors.affiliation: Diagnostic Systems Division, United States Army Medical Research Institute of Infectious Disease, Fort Detrick, MD, USA.
authors.forename: Christopher P
authors.initials: CP
authors.lastname: Stefan
authors.affiliation: Diagnostic Systems Division, United States Army Medical Research Institute of Infectious Disease, Fort Detrick, MD, USA.
authors.forename: Kitty
authors.initials: K
authors.lastname: Chase
authors.affiliation: Diagnostic Systems Division, United States Army Medical Research Institute of Infectious Disease, Fort Detrick, MD, USA.
authors.forename: Susan
authors.initials: S
authors.lastname: Coyne
authors.affiliation: Diagnostic Systems Division, United States Army Medical Research Institute of Infectious Disease, Fort Detrick, MD, USA.
authors.forename: David A
authors.initials: DA
authors.lastname: Kulesh
authors.affiliation: Diagnostic Systems Division, United States Army Medical Research Institute of Infectious Disease, Fort Detrick, MD, USA.
authors.forename: Timothy D
authors.initials: TD
authors.lastname: Minogue
authors.affiliation: Diagnostic Systems Division, United States Army Medical Research Institute of Infectious Disease, Fort Detrick, MD, USA. jeff.w.koehler.ctr@mail.mil.
authors.forename: Jeffrey W
authors.initials: JW
authors.lastname: Koehler
mesh_terms: D001117:Arenaviridae Infections / Q000175:diagnosis*; D002044:Bunyaviridae Infections / Q000175:diagnosis*; D006801:Humans; D025202:Molecular Diagnostic Techniques / Q000379:methods; D016856:Phlebovirus / Q000302:isolation & purification*; D018052:Pichinde virus / Q000302:isolation & purification*; D060888:Real-Time Polymerase Chain Reaction / Q000379:methods*; D015203:Reproducibility of Results; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods*; D012680:Sensitivity and Specificity; D013997:Time Factors; D014773:Virology / Q000379:methods*
keywords: Arenaviruses; Bunyaviruses; Pichinde virus; Punta Toro virus; Real-time RT-qPCR