Evaluation of Type-Specific Real-Time PCR Assays Using the LightCycler and J.B.A.I.D.S. for Detection of Adenoviruses in Species HAdV-C

Sporadically, HAdVs from species HAdV-C are detected in acute respiratory disease outbreaks. To rapidly type these viruses, we designed real-time PCR assays that detect and discriminate between adenovirus types HAdV-C1, -C2, -C5, and -C6. Sixteen clinical isolates from the California Department of Public Health were used to validate the new assays. Type-specific TaqMan real-time PCR assays were designed and used independently to successfully identify 16 representative specimens. The lower limit of detection for our LightCycler singleplex real-time PCR assays were calculated to be 100, 100, 100, and 50 genomic copies per reaction for HAdV-C1, HAdV-C2, HAdV-C5 and HAdV-C6, respectively. The results for the singleplex J.B.A.I.D.S. assays were similar. Our assays did not cross-react with other adenoviruses outside of species HAdV-C, respiratory syncytial virus, influenza, or respiratory disease causing bacteria. These assays have the potential to be useful as diagnostic tools for species HAdV-C infection.


Introduction
Human adenoviruses (HAdVs) were the first respiratory viruses to be isolated and characterized. Epidemiological studies show that adenoviruses are a common cause of epidemic respiratory illness in crowded adult populations [1,2]. Human adenoviruses are members of the genus Mastadenovirus and are grouped into seven species (A-G), based on their nucleic acid characteristics, penton, hexon, and fiber protein characteristics, biological properties and phylogenetic analysis [3,4,5,6,7]. They are associated with a broad range of symptoms, including acute respiratory disease (ARD), gastroenteritis, keratoconjunctivitis, and genitourinary infections [1,2].
Viruses in species HAdV-C are weakly pathogenic in adult populations [8]. In contrast, when they infect children under 2 years of age, the clinical outcome can lead to acute respiratory disease, intussusception [9,10], pneumonia, or myocarditis in infants under age 1 [11][12]. Moreover, an association between prenatal species HAdV-C infection and development of childhood acute leukemia was recently reported [13].
In the previous century, HAdVs were detected by tissue culture and discriminated by type-specific serum neutralization methods [14]. However, traditional (probeless) PCR assays have since replaced these methods owing to their greater speed, significantly lower cost, and to the decline in availability of type-specific antisera [15]. A variety of reliable PCR assays have been developed and used, including species-specific [16] and typespecific [17] tests. Universal PCR assays paired with sequence analysis has been used to provide a truly comprehensive detection and discrimination method for all HAdV types [18]. Real-time (probe-based) PCR platforms now offer even greater efficiency, improved sensitivity and specificity, and the added information value resulting from quantitative analysis of viral titers [19,20,21].
In this study we developed a series of real-time PCR assays for both the LightCycler and the military Joint Biological Agent Identification Detection System (J.B.A.I.D.S.) platforms which can detect and discriminate between HAdVs in species HAdV-C. Combined, these tools offer a rapid, high-throughput method for detection and discrimination of viruses in species HAdV-C. These assays will allow for much more rapid outbreak assessment, and, if validated as in-house diagnostic assays, more rapid individual and public health responses.

Results
LightCycler and J.B.A.I.D.S. TaqMan real-time PCR assays The detection of Human adenoviruses C1 (HAdV-C1), -C2, -C5, and -C6 by real-time PCR from cultured isolates is summarized in Table 1. All samples were tested on the LightCycler 2.0 and the J.B.A.I.D.S platforms. All positive samples were correctly identified (Table 1).

Specificity of Real-Time PCR assays
Because adenoviruses in species HAdV-C are well conserved in the hexon and fiber genes, we wanted to determine if our assays were specific to the target HAdV types. Analytical specificity data is shown in Table 3. Assays that detect HAdV-C2, -C5, and -C6 showed low level amplification with HAdV-C1, -C2, and -C5, respectively after 35 cycles. However, our assays did not react with HAdV-B3, -E4, -B7, -B11, -B14, or -B21. In addition, our realtime PCR assays did not generate false positive results when challenged with genomic DNA extracted from other agents which cause respiratory disease such as Haemophilus influenza, Influenza A virus, Human rhinovirus, Human parainfluenza virus, Human respiratory syncytial virus, Chlamydophila pneumonia, Escherichia Coli, Klebsiella pneumonia, Pseudomonas aeruginosa, Mycoplasma pneumonia, and Legionella Pneumophila.

Discussion
There has been a need for a rapid, accurate diagnostic type specific assays which are able to differentiate HAdVs. Currently there are type specific real-time PCR assays for species HAdV-B and HAdV-E [20,21], but not for species HAdV-C. Since viruses in species HAdV-C cause morbidity in people who are immunosupressed and/or immunocompromised [22,23], it is critical to be able to detect and discriminate between all common respiratory HAdV types. We describe the first series of real-time PCR assays which can discriminate between the four viruses which are known in species HAdV-C.   Since it is not possible to predict outbreaks, for example the HAdV-B14 outbreak in 2006 [24,25], it is important to have highquality assays readily available that can determine which adenovirus is present. Although the described singleplex assays can be used to determine whether or not nucleic acid from a virus in species HAdV-C is present in less than two hours, which is faster than conventional PCR, we recommend that these assays be used on isolates that are untypeable via PCR assays that have been validated for clinical samples [21].
In terms of cost, the described real-time PCR assays are less labor intensive which directly decreases the cost of testing each sample. Moreover, the cost of the primers and probes is less than a dollar per test. Thus, our assays are rapid and inexpensive.
One limitation to the described assays is that they target the hexon gene which has been shown to be incomplete when typing HAdVs [3]. If a HAdV which has the fiber of HAdV-C1 and the hexon of HAdV-C5, using the singleplex assays described in this study, the virus would be incorrectly typed as HAdV-C5. However, if these assays are used on a sample that contains two viruses from species HAdV-C, both viruses will be detected in separate capillaries.
The described singleplex assays were able to accurately detect the four known viruses in species HAdV-C. Unfortunately, after 35 cycles the primer/probe pairs for HAdV-C2, -C5, and -C6 amplified low levels of HAdV-C1, -C2, and -C5, respectively. This may be due to the fact that the viruses in species HAdV-C are genetically similar [26]. However, this was surprising in light of the genetic diversity that is present in the hexon genes of the viruses in species HAdV-C (Fig. 2). This was the region we used to design all primer/probe pairs. This demonstrates the complexity of designing type-specific assays using real-time PCR.

Conclusions
We generated single real-time PCR assays that are sensitive and specific and provide accurate quantitation of HAdV in species HAdV-C. These assays have the potential to be useful as routine diagnostic tools for the rapid detection of clinical samples on already existing platforms which are positive for HAdV-C1, -C2, -C5, and -C6.

Ethics Statement
The work reported herein was approved by the Institutional Review Board at the David Grant USAF Medical Center. Informed Consent was not required, because we did not use clinical samples.

Viral Isolates
Samples collected at CDPH were clinical isolates cultured in A549 cells, and subsequently frozen at 280uC and transported on dry ice for testing. Aliquots used in this study were subjected to one freeze/thaw cycle in the process of aliquotting upon receipt at CDPH, and one further freeze/thaw cycle when aliquotted for shipment to the David Grant Medical Center. The A549 cells used in this study were used in a previous study [5,20] and are a common cell line used for adenovirus research.  Table 3. Specificity table of each HAdV assay.

Optimization of assays
First, we performed experiments to determine the appropriate annealing temperature for each series of primers via conventional PCR. Reaction conditions were as follows: initial denaturation at 95uC for 15 min, followed by 40 cycles of denaturation at 95uC for 15 s, annealing temperature gradient ranged from 48uC to 65uC for 30 s, and extension at 72uC for 30 s. Each singleplex assay was optimized for MgCl 2 concentration as well as extension and annealing times. To optimize the MgCl 2 concentration, we used 0, 1, 2, 3, 4, 5 and 6 mM MgCl 2 as the final concentration of the PCR reaction. We also amplified a fixed amount of genomic DNA with different annealing (25, 20, and 15s) and extension times (20, 15, 10, and 5s). The FastStart High Fidelity PCR System kit was used for all conventional PCR (Roche).

Quantitative real-time PCR
The primers used to detect the different viruses in species HAdV-C are listed in Table 4 and 400 nM forward and reverse primers. Reaction conditions were as follows: initial denaturation at 95uC for 15 min, followed by 45 cycles of denaturation at 95uC for 1 s, annealing at 60uC for 15 s, and extension at 72uC for 5 s. The progress of real-time fluorescent PCR was monitored at 530 nm. To establish external standard curves for the quantification of each HAdV, genomic DNA from each strain was diluted in a 10-fold series (10 2 to 10 7 copies per reaction) and analyzed with the new assay. The samples that defined the standard curve were performed in triplicate and repeated twice.