Evaluation of rotavirus dsRNA load in specimens and body fluids from experimentally infected juvenile macaques by real-time PCR

Virology 341 (2005) 248 – 256 www.elsevier.com/locate/yviro

Evaluation of rotavirus dsRNA load in specimens and body fluids from experimentally infected juvenile macaques by real-time PCR Wei Zhao a, Mingjing Xia b, Tamika Bridges-Malveo a, Mayra Cantu´ a, Monica M. McNeal c, Anthony H. Choi c, Richard L. Ward c, Karol Sestak a,d,* a

Tulane National Primate Research Center, Covington, LA 70433, USA b Emory University School of Medicine, Atlanta, GA 30322, USA c Cincinnati Children’s Hospital Medical Center, Cincinnati, OH 45229-3039, USA d Tulane University School of Medicine, New Orleans, LA 70118, USA Received 15 March 2005; returned to author for revision 22 April 2005; accepted 30 June 2005 Available online 10 August 2005

Abstract We recently established a non-human primate model of rotavirus infection that is characterized by consistent and high levels of virus antigen shedding in stools. Here, we report that starting from post challenge day (PCD) 2, 6  103 to 1.5  106 copies of rotavirus doublestranded RNA per nanogram of total RNA were detected by real-time PCR in MA104 cells that were 48 h pre-incubated with filtered stool suspensions of three experimentally infected juvenile macaques. The peak of virus load was detected at PCD 4 – 5, followed by decreased load at PCD 6 – 11, and very low levels at PCD 12. Such a pattern corresponded to virus shedding in stools as reported recently based on enzyme-linked immunosorbent assay (ELISA) results. In addition, plasma and cerebrospinal fluids (CSF) from six infected animals were tested for the presence of rotavirus. Rotavirus extraintestinal escape was revealed in three out of six animals by a combination of real-time and nested PCR. However, very low quantities of detected viral RNA (¨20 copies/ng of total RNA) were not suggestive of viremia. Thus, the rhesus model of rotavirus infection can be exploited further in studies with vaccine candidates designed to prevent or abrogate rotavirus infection. D 2005 Elsevier Inc. All rights reserved. Keywords: Rotavirus; Rhesus; dsRNA; RT-PCR; Nested PCR; Real-time PCR

Introduction Rotaviruses along with caliciviruses are the most common etiological agents of diarrhea in infants and young children worldwide (Glass et al., 2000; Hoshino and Kapikian, 1996). Rotavirus diarrhea occurs most frequently in 6- to 36-month-old children and approximately 600,000 deaths occur each year among children in developing countries (Birch et al., 1977; Glass et al., 2004; Parashar et al., 1998, 2003; Ward et al., 2004). Several vaccine * Corresponding author. Tulane National Primate Research Center, Tulane University School of Medicine, Department of Microbiology and Immunology, 18703 Three Rivers Rd., Covington, LA 70433, USA. Fax: +1 985 871 6248. E-mail address: [email protected] (K. Sestak). 0042-6822/$ - see front matter D 2005 Elsevier Inc. All rights reserved. doi:10.1016/j.virol.2005.06.048

candidates aimed to prevent rotavirus infection have been developed but none are available for routine immunization at present (Kapikian et al., 1992; Midthun and Kapikian, 1996; Santos and Hoshino, 2005; Ward et al., 2004). In order to test the efficacy and safety of rotavirus vaccine candidates, a reliable animal model is needed. Recently, we established a non-human primate model of rotavirus infection (McNeal et al., 2005; Sestak et al., 2004). Since this model is characterized by high and consistent levels of virus shedding in stools but less consistent clinical manifestation of diarrhea, it is vitally important to be able to quantify the load of rotavirus in stools of rhesus macaques as the correlate of intestinal infection. Traditionally, in order to detect rotavirus from stool samples, methods such as ELISA, latex agglutination (LA), electron microscopy (EM), and polyacrylamide gel electro-

W. Zhao et al. / Virology 341 (2005) 248 – 256

phoresis (PAGE) have been used (Kapikian and Chanock, 1996). EM has been the diagnostic method since the discovery of the virus in 1973 (Bishop et al., 1973). The lack of sensitivity of EM is however its limitation. It was estimated that a quantity of <106 virus particles per gram of stools would likely not be detected by EM (Doane, 1994). The LA assays are used to pre-screen clinical specimens, but also lack sensitivity (Raboni et al., 2002; Sanekata et al., 1981). Because of its simplicity and sensitivity, ELISA is the method of choice in many of the diagnostic laboratories today (McNeal et al., 2005; Rabenau et al., 1998; Sestak et al., 1999). This technique is 10– 100 times more sensitive than EM, and <1 ng of purified human rotavirus can be detected using the optimized ELISA (Grauballe et al., 1981). Molecular biological techniques including hybridization, reverse transcriptase PCR (RT-PCR), and quantitative reverse transcriptase PCR (QRT-PCR) were developed as more sensitive alternatives to traditional assays for rotavirus detection (Flores et al., 1983; Gouvea et al., 1990; Rasool et al., 1993; Schwarz et al., 2002). By using RT-PCR, the detection rate of rotavirus was increased by 15 –27% (Pang et al., 1999; Simpson et al., 2003; Xu et al., 1990) and the sensitivity increased 100 times compared to ELISA (Buesa et al., 1996; Husain et al., 1995; Wilde et al., 1990). Since there are frequently compounds and/or inhibitors present in the stool samples that might diminish the efficiency of reverse transcriptase and DNA polymerase, modified protocols were established in which total or viral RNA is extracted from rotavirus-infected cells previously cultured in vitro with filtered stool fluids instead of directly from stools (Buesa et al., 1996; Kim et al., 2002). Competitive PCR is a commonly used quantitative method (Piatak et al., 1993; Scadden et al., 1992); however, this technique is labor intensive and is only semi-quantitative (Ferre, 1992; Sestak et al., 2003). The more recently developed real-time PCR represents a technological advance in the field of molecular diagnostics, characterized by high accuracy, sensitivity, and reproducibility (Bustin, 2000). It was demonstrated that as few as 10 copies of RNA can be amplified by real-time PCR (Schwarz et al., 2002). Pang et al. (2004b) reported the detection of rotavirus in stool samples diluted as much as 10 9 using real-time PCR. As of today, data regarding the use of real-time PCR for quantification of rotavirus are few and limited to human clinical specimens (Kang et al., 2004; Kurokawa et al., 2004; Pang et al., 2004b). Since rotavirus is shed in large quantities during acute infection (Midthun and Kapikian, 1996), it is valuable to quantify fecal shedding as the correlate of infection. In this study, we used real-time PCR to measure rotavirus load in samples from experimentally infected macaques. The rotavirus strain we used (TUCH) was recently described in detail elsewhere (McNeal et al., 2005). In addition, we used real-time PCR in conjunction with nested PCR to examine the CSF and plasma samples collected from

249

juvenile macaques after a detectable rotavirus infection in order to investigate the possibility of rotavirus extraintestinal escape (Blutt et al., 2003; Mossel and Ramig, 2002; Ramig, 2004).

Results Design of RT-PCR-based protocols for detecting and quantifying TUCH rotavirus Total RNA was isolated from cultured MA104 cells previously pre-incubated with filtered suspensions of tested samples. TUCH rotavirus VP6 gene-specific primers were used to detect and quantify rotavirus in tested samples by first-round or nested PCR, in combination with real-time PCR. For those samples in which rotavirus was undetectable by first-round PCR, nested PCR was further employed in conjunction with real-time PCR. TUCH rotavirus VP6 outer primers were used in the first-round PCR, which amplified a 489-bp sequence in the VP6 gene (nt 600 – 1089), and the VP6 inner primers were used in nested PCR and real-time PCR, which amplified a 140-bp sequence in the VP6 gene (nt 874– 1014). DNA sequencing was used to verify the specificity of first-round and nested PCR amplification products: 99% identities were found between the TUCH VP6 gene and PCR products (data not shown). Quantification of TUCH rotavirus by real-time PCR Generation of standards The glyceraldehyde-3-phosphate dehydrogenase (GAPDH) gene was used as the external standard of known DNA concentration to extrapolate the VP6 gene copy numbers. It was serially (10-fold) diluted in a range from 4.2  107 to 4.2  102 copies per reaction. The GAPDH forward and reverse primers were used to generate the 108 base pair product. The GAPDH standard curve revealed an excellent correlation between the Ct value and GAPDH copy numbers (r = 0.999). The melting curve of the GAPDH standard showed a single sharp peak at the temperature of 81.8 -C, while the non-template control (NTC) showed no significant fluorescent signal (not shown), indicating a high specificity of amplification for the GAPDH standard in real-time PCR. The standard curve was used for enumeration of rotavirus copy numbers in all of the tested samples. Quantification of TUCH rotavirus in stool samples A total of 48 stool samples collected from three juvenile rhesus macaques were analyzed by real-time PCR. In order to avoid highly variable results due to the presence of RNA inhibitors in stools, total RNA was extracted from MA104 cells that were pre-incubated with filtered sample suspensions for 48 h rather than directly from stools. The primers amplified a 140-bp (nested PCR site) sequence in the TUCH VP6 gene, similar in length to that of the GAPDH standard

250

W. Zhao et al. / Virology 341 (2005) 248 – 256

(108 bp). Amplification plots generated with samples from each of the three macaques revealed a wide range of viral loads present in analyzed specimens (Fig. 1A). The specificity of the real-time PCR for all the samples was verified by melting curves (Fig. 1B). The single sharp peak of each melting curve and the identical melting temperature (80.6 -C) corresponding to multiple rotavirus-positive samples and the TUCH rotavirus positive control suggested that the amplification was rotavirus-specific. Additional peaks, that would have indicated the presence of nonspecific amplification, were not observed in any of the samples including the TUCH rotavirus positive control. The real-time PCR was able to detect changes in copy numbers among samples collected at different PCDs (Fig. 2A). The results revealed high average levels of viral RNA copies starting from PCD 2 (3.8  105 copies/ng of extracted total RNA) contained in total RNA obtained from MA104 cells that were pre-incubated with the filtered stool suspensions from all three macaques. The peak of viral shedding extrapolated by this method occurred at PCD 4 – 5, followed by decreased shedding at PCD 6 –11, and very low levels at PCD 12 (Fig. 2A). At the level of 20 copies, the differences between the negative and positive samples could

no longer be reliably determined without the help of additional tools such as nested PCR or DNA sequencing. Therefore, the assay cutoff was determined at the level of 20 copies/ng of total RNA. The melting curve characteristics of the real-time PCR indicated that temperature peaks of both TUCH and GAPDH genes were located within the 81 T 1 -C interval. These results were corroborated by two independent realtime PCR tests for each sample. In addition, the absence of genomic DNA in RNA samples was verified by comparing the real-time PCR cycles with and without reverse transcriptase. No rotavirus-specific product was detected in samples without reverse transcriptase. Corroboration of TUCH rotavirus load in stools by ELISA The ELISA values corresponding to rotavirus rectal shedding by three macaques (FB82, FB88, and FB97) were used in our study to validate results generated by real-time PCR (Fig. 2A). These values were individually reported elsewhere (McNeal et al., 2005). In our study, averages T SEM (standard error mean) at each time point corresponding to 3 specimens from the 3 above animals were used to compare the ELISA viral load data with that of real-time PCR. A positive correlation (r = 0.78, P < 0.001) was found between the real-time PCR and ELISA results (Fig. 2B). Reproducibility of results generated by our real-time PCR was evaluated by multiple (8) testing of five samples of different viral loads including a negative control. Variability of resultant viral loads did not surpass 3  SEM for any of the 5 sets (Fig. 2C). Sensitivity of first-round, nested and real-time PCR

Fig. 1. (A) Amplification plots (fluorescence signal versus cycle number) reflecting the different loads of TUCH rotavirus corresponding with 16 stool samples collected at PCD 0 – 15 from macaque #2. During the initial cycles of amplification reaction, no or a very small change in fluorescence signal above the baseline ( y = 0) is observed. An increase in fluorescence above the baseline indicates the detection of accumulated PCR product. (B) Melting curves (x = temperature, y = changes in density of fluorescence signal) of the stool samples. Each sample produced a single sharp peak, and all of them overlapped and showed the same melting temperature (80.6 -C).

Based on the GAPDH standard, the real-time PCR sensitivity threshold was set at the level of 20 copies/ng of extracted total RNA. Less than 20 copies/ng RNA yielded real-time PCR signals that were not distinguishable from negative samples. When comparing this sensitivity with sensitivities of first-round and nested PCR, we found that nested PCR was the most sensitive. At least 10 and 1000 times higher sensitivity of nested PCR than that of realtime and first-round PCR was found, respectively. Thus, deployment of real-time PCR in conjunction with nested PCR was used in a few selected cases (CSF and plasma sample) in which low (20 copies) amounts of TUCH rotavirus were detected. In addition, if such a screening resulted in positive nested PCR amplification, DNA sequencing was used to confirm the authenticity of the amplification product. Detection of rotavirus in plasma and cerebrospinal fluids (CSF) In order to investigate the possible spread and/or escape of rotavirus outside the gastrointestinal tract in our juvenile

W. Zhao et al. / Virology 341 (2005) 248 – 256

251

Fig. 2. Stool samples that were collected from three TUCH rotavirus-infected juvenile macaques were tested by both ELISA and real-time PCR assays to approximate the load of rotavirus in stools. (A) ELISA values reflect the amount (ng per ml of 20% stool suspension) of rotavirus antigen while real-time PCR values reflect the number of rotavirus VP6 copies per ng of total RNA that was extracted from MA104 cells. Mean and standard error bars were calculated for each time point based on values corresponding to 3 samples from 3 animals. (B) Correlation between the real-time PCR and ELISA was evaluated by Spearman’s test ( P < 0.001, r = 0.78). (C) Five samples of different viral loads including the negative control were each amplified in eight independent MA104 cultures and tested by real-time PCR to verify the assay’s reproducibility. For each of the 5 sets, variability of resultant viral loads did not surpass 3  SEM.

rhesus macaque model of rotavirus infection, we examined the CSF and plasma samples collected at PCD 7 after a detectable TUCH rotavirus infection from six animals. The results of real-time PCR showed very few copies (20) of virus in samples from all six animals. Since these values were either at the limit or below the assay’s sensitivity cutoff value, nested PCR was employed. Two plasma samples and one CSF sample from three out of six animals showed positive results by nested PCR (Fig. 3). DNA sequencing analysis confirmed that the nested PCR products were TUCH rotavirus VP6 gene specific (>99% sequence

Fig. 3. Detection of rotavirus in plasma and cerebrospinal fluids (CSF) by nested RT-PCR. One Al of cDNA prepared from plasma or CSF samples collected at PCD 7 from six TUCH rotavirus-infected macaques was used as the template of first-round PCR, and 1 Al of the first-round PCR products was used as the template for nested PCR. Lanes 1 – 6—plasma# 1 – 6, lanes 7 – 12—CSF# 1 – 6, lanes 13 – 14—negative controls. In two plasma samples (lanes 1 and 4) and one cerebrospinal fluid sample (lane 12), rotavirus-specific RNA was detected by nested PCR. The TUCH rotavirus specificity of all three amplification products was confirmed by DNA sequencing (>99% homology).

homology), indicating the TUCH rotavirus in extraintestinal samples of juvenile rhesus macaques, although in very low quantities.

Discussion Based on the TUCH rotavirus VP6 gene, which encodes the most abundant protein of the virus, we implemented a sensitive quantitative RT-PCR system, capable of detecting and quantifying TUCH rotavirus RNA. In order to accomplish this with stool specimens that are known to contain high levels of RNA degrading enzymes and other components that might interfere with PCR, we implemented a method where filtered stool suspensions were preincubated with rotavirus-susceptible cells (MA104) for 48 h prior to extraction of total RNA from these cells. This approach was successfully used in other studies with PCR amplification of feces-borne RNA viruses (Buesa et al., 1996; Kim et al., 2002). To generate cDNA, we chose the random priming method instead of target-specific synthesis due to the following reasons: Target-specific primers synthesize specific cDNA, but require separate priming reactions for each target, hence it is not possible to return to the same preparation and amplify other targets at a later stage. Thus, it might not be practical if only limited amounts of RNA are available. Although random primers generate both non-

252

W. Zhao et al. / Virology 341 (2005) 248 – 256

specific and specific cDNA transcripts, they also yield more cDNA (Bustin and Nolan, 2004; Iturriza-Gomara et al., 1999). There have been several reports that real-time PCR works as well with a DNA-binding dye as it does with fluorescent sequence-specific probes (Hein et al., 2001; Ramos-Payan et al., 2003). We chose the SYBR green I probe in this study because this approach allows the detection of DNA generated during PCR (Morrison et al., 1998), and it can be incorporated into established protocols that use legacy primers and experimental conditions (Bustin and Nolan, 2004). Utilizing the real-time PCR technology, we detected and further evaluated rotavirus RNA load in proportion to total RNA from cells pre-incubated with filtered stools collected from three TUCH rotavirus-infected rhesus macaques. Large quantities of rotavirus shed in feces of all three macaques between PCD 4 and 5 were detected by this method. These results were consistent with those of ELISA reported elsewhere (McNeal et al., 2005; Sestak et al., 2004). The reason why the correlation coefficient between the two methods was only moderate (r = 0.78) was possibly due to more rapid amplification of TUCH rotavirus in cases of samples with high viral loads than these with low viral loads during 48 h of virus pre-incubation with MA104 cells. We hypothesize that in cases of human rotavirus strains that are capable of infecting simian MA104 cells but are not amplifying as aggressively as TUCH rotavirus, this correlation would be higher. In such instances, however, relevant PCR primers would have to be utilized. Also, a shorter period (12 – 24 instead of 48 h) of sample pre-amplification on MA104 cells could improve the correlation with ELISA. To maintain the consistent conditions in this study, the incubation time of all samples with MA104 cells were kept 48 h. No cytopathic effect, cell lysis, and/or release of viral particles into cell-culture media were observed (Supplementary data). Limitations of this quantification system are (a) the virus must maintain its infectivity in order to enter the MA104 cells and (b) an imperfect correlation with direct quantification (ELISA). If consistent and high yields of sufficiently pure RNA could be achieved directly from stool samples, the above limitations could likely be avoided. Our protocol utilized the GAPDH house-keeping gene as an external standard and SYBR green I dye to label the amplification products. SYBR Green I is able to bind the double-stranded DNA products in real-time during the amplification reaction (Giulietti et al., 2001; Poddar, 2004; Xu and Miller, 2004). The sensitivity of our real-time PCR was estimated at the level of 20 copies/ng of total RNA that was 102 times more sensitive than first-round PCR. Such sensitivity is in agreement with results reported by Pang et al. (2004a). When performed in conjunction with nested PCR, our real-time PCR system could detect even a single copy of rotavirus RNA in 1 ng of total RNA. Based on reports of rotavirus extraintestinal escape and suggested viremia in children (Blutt et al., 2003; Kawa-

shima et al., 2004; Mossel and Ramig, 2002; Ramig, 2004), we decided to test the serum and CSF samples collected from six juvenile macaques at the acute stage of rotavirus infection for the presence of rotavirus-specific RNA. We were interested to determine (a) if rotavirus can be detected in extraintestinal sites of infected juvenile macaques and, if so, (b) in what quantities. In order to assure the accuracy and sensitivity of our detection system, we performed real-time PCR in conjunction with nested PCR. In our detection system, nested PCR was at least 103 times more sensitive than first-round PCR, consistent with findings of Yamakawa et al. (1995). Using the combination of nested PCR and real-time PCR, we detected the TUCH rotavirus VP6 gene in very low copy numbers (20 copies/ng RNA) in three out of six rotavirus-infected animals (two plasma samples and one CSF sample). Despite the very low copy number, we confirmed by DNA sequencing that these nested PCR products were TUCH rotavirus VP6 gene-specific. Thus, our results corroborated the results of others that rotavirus might escape outside the gastrointestinal tract (Blutt et al., 2003; Mossel and Ramig, 2002; Nishimura et al., 1993; Ushijima et al., 1994). Moreover, our study is the first to report the escape of rotavirus into extraintestinal tissues of any nonhuman primate. Interestingly, in one macaque, we were able to reveal the presence of TUCH rotavirus RNA in an immunoprivileged site, i.e., the central nervous system. This finding might support the clinical observations of Kawashima et al. (2004) who suggested the link between the rotavirus-induced gastroenteritis and encephalopathy in some Japanese children. Nevertheless, further pathogenesis studies need to be conducted in order to fully elucidate the significance of the observed rotavirus extraintestinal escape. In summary, we report the development and application of a new, sensitive molecular technique for detection of TUCH rotavirus in samples from non-human primates. Our real-time PCR can be used as a quantitative tool in studies with rotavirus vaccine candidates where the main objective is to demonstrate the reduction or complete abrogation of intestinal rotavirus infection. Moreover, our nested PCR combined with real-time PCR can be utilized in pathogenesis studies to track the low quantities of virus in extraintestinal tissues.

Materials and methods Rhesus macaques and sample collection Stool, plasma, and cerebrospinal fluid (CSF) samples were collected from six rotavirus-infected juvenile rhesus macaques (Macaca mulatta, FB82, 83, 88, 91, 97, and FC06). Animals derived by cesarean sections were infected orally with TUCH rotavirus at the age of 3 weeks and kept in biosafety level 2 (BL2) in accordance with standards of

W. Zhao et al. / Virology 341 (2005) 248 – 256

the Guide for the Care and Use of Laboratory Animals and the Association for Assessment and Accreditation of Laboratory Animal Care as described in detail elsewhere (McNeal et al., 2005; Sestak et al., 2004). Stools were collected daily up to 4 weeks post challenge and frozen at 80 -C upon collection. Five hundred microliters of plasma and/or CSF was collected on the PCD 7 and stored at 80 -C. Pre-amplification of rotavirus-suspect samples on MA104 cells

253

mixture: 2.5 Al of 5 reaction buffer (250 mM Tris –HCl [pH 8.3], 375 mM KCl, 15 mM MgCl2, and 50 mM DTT), 100 units of M-MLV Reverse Transcriptase (Promega, Madison, WI), 2.5 Al of dNTP containing 10 mM of dATP, dCTP, dGTP, and dTTP (SIGMA, St. Louis, MO), 12.5 units of RNasin Ribonuclease Inhibitor (Promega, Madison, WI), and nuclease-free water in a final volume of 12.5 Al. The reaction mixture was incubated at 37 -C for 60 min followed by incubation at 70 -C for 10 min to inactivate the M-MLV Reverse Transcriptase. Primers

Stool specimens were thawed at room temperature, weighed, suspended in 10% (w/v) phosphate-buffered saline (PBS; pH 7.4), and centrifuged for 10 min, at 57g, 4 -C to remove the debris. Liquid supernatants were filtered through a 40-Am pore size filter (Fisher Scientific Company, Pittsburgh, PA). Bacteria-free supernatants were diluted (30% v/v) with Eagle’s Minimum Essential Media (MEM, ATCC, Manassas, VA) and 500 Al of suspension was incubated with 25 Ag/ml of trypsin-EDTA (Cat#15400-054, Gibco, Carlsbad, CA) at 37 -C for 30 min, then added to simian epithelial cell line MA104 (ATCC) monolayers previously grown to confluence in 25 cm2 cell culture flasks (Fisher Scientific, Pittsburgh, PA). After incubation (37 -C, 60 min, slow rocking), the inoculum was removed and 5 ml of MEM was added to each flask. Flasks were incubated at 37 -C with 5% CO2 for 48 h. For plasma and CSF samples, 500 Al of trypsin-treated, 30% MEM (v/v) sterile sample suspensions were used to infect the MA104 cells as described above for stool samples. Isolation of total RNA After 48 h of incubation with MA104 cells, the total RNA was extracted from harvested cells using the Trireagent (Molecular Research Center, Cincinnati, OH) according to the manufacturer’s instructions. Equal volumes of MEM and cell-culture adapted TUCH rotavirus (104 fluorescein focus units per milliliter) were used as negative and positive controls, respectively. Extracted total RNA was quantified by spectrophotometry at 260 nm and stored at 80 -C. RNA integrity was determined visually using 1.0% denaturing agarose gels with 1 MOPS (Sigma, St. Louis, MO) running buffer according to standard protocols (Sambrook et al., 1989).

TUCH-rotavirus-specific primers Two pairs of rotavirus-specific primers were designed based on TUCH rotavirus VP6 gene sequence (GenBank accession number AY594670) using the Seqweb version 2 software (Accelrys, San Diego, CA). The forward outer primer (5V-CGCTCCAGCCAATACACAAC-3V) and the reverse outer primer (5V-CGGTGGAAAAACAGGTCCAAC-3V) that amplified a 489-bp fragment (nt 600– 1089) of the TUCH VP6 gene were used in first-round PCR. The forward inner primer (5V-TTCCAATTGTTGCGCCCAC-3V) and reverse inner primer (5V-AGCATCAGCAAGTACTGATTCG-3V) that amplified a 140-bp fragment (nt 874 – 1014) of the TUCH VP6 gene were used in nestedPCR and real-time PCR. The primers were synthesized by Integrated DNA Technologies. These primers were TUCHrotavirus-specific. Only 86% sequence identities were found with other group A rotaviruses based on BLASTN searches in GenBank database (Table 1). Primers for human glyceraldehyde-3-phosphate dehydrogenase (GAPDH) house-keeping control gene The primers for GAPDH gene were designed based on Homo sapiens GAPDH sequence (GenBank accession number BC023632) using the Seqweb software, version 2 (Accelrys). The forward primer (5V-ACTCCTCCACCTTTGACGCTG-3V) and reverse primer (5V-GGTCCACCACCCTGTTGCTG-3V) amplified a 108-bp fragment of the GAPDH gene. The primers were synthesized by Integrated DNA Technologies. The GAPDH-based standard curve was used for enumeration of rotavirus copy numbers in all of the tested samples. First-round and nested PCR

cDNA synthesis Reverse transcription was performed with 1 Ag of total RNA and 0.25 Ag of random hexamers (Integrated DNA Technologies, Coralville, IA) in a total volume of 5.5 Al, at 70 -C for 5 min to melt any secondary structures within the template. Following the reverse transcription, reaction mixture was cooled immediately on ice for 2 min and the following components were added to the primer/template

The TUCH VP6 outer primers were used in the firstround PCR. One Al of 1:80 ddH2O-diluted cDNA was used as the template (equivalent to 1 ng of total RNA). The firstround PCR was performed at 95 -C for 3 min, followed by 30 cycles of 95 -C for 1 min, 55 -C for 1 min, 72 -C for 2 min, and a final extension step of 72 -C for 10 min using the 16-well thermocycler (MJ Research, Watertown, MA). Nested PCR was carried out under the same conditions,

254

W. Zhao et al. / Virology 341 (2005) 248 – 256

Table 1 Comparison of nucleotide identities of the TUCH rotavirus VP6 genespecific real-time PCR product with counterparts of 21 other group A rotaviruses Group A rotavirus

Identity [%] with TUCH simian rotavirusa

GenBank accession number

IS2 (human) US1205 (human) TB-Chen (human) 97VB53 (human) E210 (human) 116E (human) CJN (human) Wa (human) SA11 (simian) BRV033 (porcine) B223 (porcine) CRW-8 (porcine) NCDV (bovine) 22R (bovine) WC3 (bovine) H-2 (equine) FI-14 (equine) LP14 (ovine) EDIM (murine) EW (murine) EmcN (murine)

86 86 81 81 80 78 77 78 80 81 80 79 85 85 81 80 80 84 78 78 78

X94617 AF079357 AY787645 AF260931 U36240 U85998 AF461757 K02086 AY187029 AF317126 AF317128 U82971 AF317127 AB040055 AF411322 D00324 D00323 L11595 U65988 U36474 AY267007

a

Sequence identities were determined based on BLASTN searches in GenBank database.

but with TUCH VP6 inner primers, and the template was 1 Al of first-round PCR product. The PCR products were visualized with ethidium-bromide after electrophoretic separation in 2.0% agarose gels according to standard protocols (Sambrook et al., 1989) and the images were recorded using the Alpha Imager 2000 Documentation and Analysis System (Alpha Innotech, San Leandro, CA).

(http://www.appliedbiosystems.com/support/software/ 7700pdates.cfm, ABI). Quantification of TUCH rotavirus in stool samples using the ELISA The subsets of previously reported (McNeal et al., 2005) ELISA values associated with the presence of TUCH rotavirus in stools of our three rhesus macaques (FB82, FB88 and FB97) were used to calculate the group means and standard error means with the objective to compare these results with real-time PCR (Fig. 2). Statistical evaluation of correlation between real-time PCR and ELISA The correlation between the real-time PCR and ELISA viral loads was evaluated by Spearman’s test.

Acknowledgments W. Zhao and M. Xia contributed equally to this study. Support was provided by NIAID grant AI056847-01 (A.C.) and by TNPRC grant RR00164. The excellent technical assistance of Gloria Jackson, Drs. Pyone Pyone Aye, Xavier Alvarez, Mahesh Mohan, Marion Ratterree, and Erin Ribka is greatly appreciated. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at doi:10.1016/j.virol.2005. 06.048.

Quantification of TUCH rotavirus by real-time PCR Real-time PCR was carried out using the ABI Prism 7700 Sequence Detection System (Applied Biosystems, Foster City, CA). Each PCR reaction included 10 Al of 2 master mix (DyNAmo HS SYBR Green qPCR Kit, MJ research, Waltham, MA). It contained modified DNA polymerase, SYBR Green I, PCR buffer, 5 mM MgCl2, dNTP mix including dUTP, 0.5 AM of TUCH VP6 inner or GAPDH primers, 1  ROX reference dye, cDNA (equivalent to 1 ng of total RNA) from the RT reaction. After heating each sample to 95 -C for 15 min, it was followed by 40 cycles consisting of 94 -C for 10 s, 55 -C for 30 s, and 72 -C for 30 s. All tested samples were run in duplicate. Two negative controls were used for each run. The quantity of the TUCH rotavirus VP6 gene was deduced from the regression line and translated into copy number per nanogram of total RNA by using the SDS 1.9.1 Software (URL: http://www.appliedbiosystems.com/support/software/ 7700pdates.cfm, Applied Biosystems). The melting curves were obtained with ABI Prism 7700 software, version 1.7

References Birch, C.J., Lewis, F.A., Kennett, M.L., Homola, M., Pritchard, H., Gust, I.D., 1977. A study of the prevalence of rotavirus infection in children with gastroenteritis admitted to an infectious diseases hospital. J. Med. Virol. 1 (1), 69 – 77. Bishop, R.F., Davidson, G.P., Holmes, I.H., Ruck, B.J., 1973. Virus particles in epithelial cells of duodenal mucosa from children with acute non-bacterial gastroenteritis. Lancet 2 (7841), 1281 – 1283. Blutt, S.E., Kirkwood, C.D., Parreno, V., Warfield, K.L., Ciarlet, M., Estes, M.K., Bok, K., Bishop, R.F., Conner, M.E., 2003. Rotavirus antigenaemia and viraemia: a common event? Lancet 362 (9394), 1445 – 1449. Buesa, J., Colomina, J., Raga, J., Villanueva, A., Prat, J., 1996. Evaluation of reverse transcription and polymerase chain reaction (RT/PCR) for the detection of rotaviruses: applications of the assay. Res. Virol. 147 (6), 353 – 361. Bustin, S.A., 2000. Absolute quantification of mRNA using real-time reverse transcription polymerase chain reaction assays. J. Mol. Endocrinol. 25 (2), 169 – 193. Bustin, S.A., Nolan, T., 2004. Pitfalls of quantitative real-time reversetranscription polymerase chain reaction. J. Biomol. Tech. 15 (3), 155 – 166.

W. Zhao et al. / Virology 341 (2005) 248 – 256 Doane, F.W., 1994. Electron microscopy for the detection of gastroenteritis viruses. In: Kapikian, A.Z. (Ed.), Viral Infections of the Gastrointestinal Tract. Marcel Dekker, New York, NY, pp. 101 – 130. Ferre, F., 1992. Quantitative or semi-quantitative PCR: reality versus myth. PCR Methods Appl. 2 (1), 1 – 9. Flores, J., Boeggeman, E., Purcell, R.H., Sereno, M., Perez, I., White, L., Wyatt, R.G., Chanock, R.M., Kapikian, A.Z., 1983. A dot hybridisation assay for detection of rotavirus. Lancet 1 (8324), 555 – 558. Giulietti, A., Overbergh, L., Valckx, D., Decallonne, B., Bouillon, R., Mathieu, C., 2001. An overview of real-time quantitative PCR: applications to quantify cytokine gene expression. Methods 25 (4), 386 – 401. Glass, R.I., Noel, J., Ando, T., Fankhauser, R., Belliot, G., Mounts, A., Parashar, U.D., Bresee, J.S., Monroe, S.S., 2000. The epidemiology of enteric caliciviruses from humans: a reassessment using new diagnostics. J. Infect Dis. 181 (Suppl. 2), S254 – S261. Glass, R.I., Bresee, J.S., Parashar, U.D., Jiang, B., Gentsch, J., 2004. The future of rotavirus vaccines: a major setback leads to new opportunities. Lancet 363 (9420), 1547 – 1550. Gouvea, V., Glass, R.I., Woods, P., Taniguchi, K., Clark, H.F., Forrester, B., Fang, Z.Y., 1990. Polymerase chain reaction amplification and typing of rotavirus nucleic acid from stool specimens. J. Clin. Microbiol. 28 (2), 276 – 282. Grauballe, P.C., Vestergaard, B.F., Meyling, A., Genner, J., 1981. Optimized enzyme-linked immunosorbent assay for detection of human and bovine rotavirus in stools: comparison with electronmicroscopy, immunoelectro-osmophoresis, and fluorescent antibody techniques. J. Med. Virol. 7 (1), 29 – 40. Hein, J., Schellenberg, U., Bein, G., Hackstein, H., 2001. Quantification of murine IFN-gamma mRNA and protein expression: impact of real-time kinetic RT-PCR using SYBR green I dye. Scand. J. Immunol. 54 (3), 285 – 291. Hoshino, Y., Kapikian, A.Z., 1996. Classification of rotavirus VP4 and VP7 serotypes. Arch. Virol., Suppl. 12, 99 – 111. Husain, M., Seth, P., Broor, S., 1995. Detection of group A rotavirus by reverse transcriptase and polymerase chain reaction in feces from children with acute gastroenteritis. Arch. Virol. 140 (7), 1225 – 1233. Iturriza-Gomara, M., Green, J., Brown, D.W., Desselberger, U., Gray, J.J., 1999. Comparison of specific and random priming in the reverse transcriptase polymerase chain reaction for genotyping group A rotaviruses. J. Virol. Methods 78 (1 – 2), 93 – 103. Kang, G., Iturriza-Gomara, M., Wheeler, J.G., Crystal, P., Monica, B., Ramani, S., Primrose, B., Moses, P.D., Gallimore, C.I., Brown, D.W., Gray, J., 2004. Quantitation of group A rotavirus by real-time reversetranscription-polymerase chain reaction: correlation with clinical severity in children in South India. J. Med. Virol. 73 (1), 118 – 122. Kapikian, A.Z., Chanock, R.M., 1996. Rotaviruses. In: Fields, B.N., Knipe, D.M., Howley, P.M. (Eds.), Fields Virol., vol. 2, 3rd ed. LippincottRaven Press, Philadelphia, PA. Kapikian, A.Z., Vesikari, T., Ruuska, T., Madore, H.P., Christy, C., Dolin, R., Flores, J., Green, K.Y., Davidson, B.L., Gorziglia, M., et al., 1992. An update on the ‘‘Jennerian’’ and modified ‘‘Jennerian’’ approach to vaccination of infants and young children against rotavirus diarrhea. Adv. Exp. Med. Biol. 327, 59 – 69. Kawashima, H., Inage, Y., Ogihara, M., Kashiwagi, Y., Takekuma, K., Hoshika, A., Mori, T., Watanabe, Y., 2004. Serum and cerebrospinal fluid nitrite/nitrate levels in patients with rotavirus gastroenteritis induced convulsion. Life Sci. 74 (11), 1397 – 1405. Kim, K., Park, J., Chung, Y., Cheon, D., Lee, I.B., Lee, S., Yoon, J., Cho, H., Song, C., Lee, K.H., 2002. Use of internal standard RNA molecules for the RT-PCR amplification of the faeces-borne RNA viruses. J. Virol. Methods 104 (2), 107 – 115. Kurokawa, M., Ono, K., Nukina, M., Itoh, M., Thapa, U., Rai, S.K., 2004. Detection of diarrheagenic viruses from diarrheal fecal samples collected from children in Kathmandu, Nepal. Nepal Med. Coll. J. 6 (1), 17 – 23. McNeal, M.M., Sestak, K., Choi, A.H., Basu, M., Cole, M.J., Aye, P.P.,

255

Bohm, R.P., Ward, R.L., 2005. Development of a rotavirus-shedding model in rhesus macaques, using a homologous wild-type rotavirus of a new P genotype. J. Virol. 79 (2), 944 – 954. Midthun, K., Kapikian, A.Z., 1996. Rotavirus vaccines: an overview. Clin. Microbiol. Rev. 9 (3), 423 – 434. Morrison, T.B., Weis, J.J., Wittwer, C.T., 1998. Quantification of low-copy transcripts by continuous SYBR Green I monitoring during amplification. Biotechniques 24 (6), 954 – 958, 960, 962. Mossel, E.C., Ramig, R.F., 2002. Rotavirus genome segment 7 (NSP3) is a determinant of extraintestinal spread in the neonatal mouse. J. Virol. 76 (13), 6502 – 6509. Nishimura, S., Ushijima, H., Shiraishi, H., Kanazawa, C., Abe, T., Kaneko, K., Fukuyama, Y., 1993. Detection of rotavirus in cerebrospinal fluid and blood of patients with convulsions and gastroenteritis by means of the reverse transcription polymerase chain reaction. Brain Dev. 15 (6), 457 – 459. Pang, X.L., Joensuu, J., Hoshino, Y., Kapikian, A.Z., Vesikari, T., 1999. Rotaviruses detected by reverse transcription polymerase chain reaction in acute gastroenteritis during a trial of rhesus-human reassortant rotavirus tetravalent vaccine: implications for vaccine efficacy analysis. J. Clin. Virol. 13 (1 – 2), 9 – 16. Pang, X., Lee, B., Chui, L., Preiksaitis, J.K., Monroe, S.S., 2004a. Evaluation and validation of real-time reverse transcription-PCR assay using the lightCycler system for detection and quantitation of norovirus. J. Clin. Microbiol. 42 (10), 4679 – 4685. Pang, X.L., Lee, B., Boroumand, N., Leblanc, B., Preiksaitis, J.K., Yu Ip, C.C., 2004b. Increased detection of rotavirus using a real time reverse transcription-polymerase chain reaction (RT-PCR) assay in stool specimens from children with diarrhea. J. Med. Virol. 72 (3), 496 – 501. Parashar, U.D., Bresee, J.S., Gentsch, J.R., Glass, R.I., 1998. Rotavirus. Emerg. Infect. Dis. 4 (4), 561 – 570. Parashar, U.D., Hummelman, E.G., Bresee, J.S., Miller, M.A., Glass, R.I., 2003. Global illness and deaths caused by rotavirus disease in children. Emerg. Infect. Dis. 9 (5), 565 – 572. Piatak Jr., M., Saag, M.S., Yang, L.C., Clark, S.J., Kappes, J.C., Luk, K.C., Hahn, B.H., Shaw, G.M., Lifson, J.D., 1993. High levels of HIV-1 in plasma during all stages of infection determined by competitive PCR. Science 259 (5102), 1749 – 1754. Poddar, S.K., 2004. Differential detection of B. pertussis from B. parapertussis using a polymerase chain reaction (PCR) in presence of SYBR green1 and amplicon melting analysis. Mol. Cell. Probes 18 (6), 429 – 435. Rabenau, H., Knoll, B., Allwinn, R., Doerr, H.W., Weber, B., 1998. Improvement of the specificity of enzyme immunoassays for the detection of rotavirus and adenovirus in fecal specimens. Intervirology 41 (2 – 3), 55 – 62. Raboni, S.M., Nogueira, M.B., Hakim, V.M., Torrecilha, V.T., Lerner, H., Tsuchiya, L.R., 2002. Comparison of latex agglutination with enzyme immunoassay for detection of rotavirus in fecal specimens. Am. J. Clin. Pathol. 117 (3), 392 – 394. Ramig, R.F., 2004. Pathogenesis of intestinal and systemic rotavirus infection. J. Virol. 78 (19), 10213 – 10220. Ramos-Payan, R., Aguilar-Medina, M., Estrada-Parra, S., Gonzalez, Y.M.J.A., Favila-Castillo, L., Monroy-Ostria, A., Estrada-Garcia, I.C., 2003. Quantification of cytokine gene expression using an economical real-time polymerase chain reaction method based on SYBR Green I. Scand. J. Immunol. 57 (5), 439 – 445. Rasool, N.B., Larralde, G., Gorziglia, M.I., 1993. Determination of human rotavirus VP4 using serotype-specific cDNA probes. Arch. Virol. 133 (3 – 4), 275 – 282. Sambrook, J., Fritsch, E.F., Maniatis, T., 1989. Molecular cloning: A Laboratory Manual, 2nd edR Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY. Sanekata, T., Yoshida, Y., Okada, H., 1981. Detection of rotavirus in faeces by latex agglutination. J. Immunol. Methods 41 (3), 377 – 385. Santos, N., Hoshino, Y., 2005. Global distribution of rotavirus serotypes/

256

W. Zhao et al. / Virology 341 (2005) 248 – 256

genotypes and its implication for the development and implementation of an effective rotavirus vaccine. Rev. Med. Virol. 15 (1), 29 – 56. Scadden, D.T., Wang, Z., Groopman, J.E., 1992. Quantitation of plasma human immunodeficiency virus type 1 RNA by competitive polymerase chain reaction. J. Infect. Dis. 165 (6), 1119 – 1123. Schwarz, B.A., Bange, R., Vahlenkamp, T.W., Johne, R., Muller, H., 2002. Detection and quantitation of group A rotaviruses by competitive and real-time reverse transcription-polymerase chain reaction. J. Virol. Methods 105 (2), 277 – 285. Sestak, K., Zhou, Z., Shoup, D.I., Saif, L.J., 1999. Evaluation of the baculovirus-expressed S glycoprotein of transmissible gastroenteritis virus (TGEV) as antigen in a competition ELISA to differentiate porcine respiratory coronavirus from TGEV antibodies in pigs. J. Vet. Diagn. Invest. 11 (3), 205 – 214. Sestak, K., Aye, P.P., Buckholt, M., Mansfield, K.G., Lackner, A.A., Tzipori, S., 2003. Quantitative evaluation of Enterocytozoon bieneusi infection in simian immunodeficiency virus-infected rhesus monkeys. J. Med. Primatol. 32 (2), 74 – 81. Sestak, K., McNeal, M.M., Choi, A., Cole, M.J., Ramesh, G., Alvarez, X., Aye, P.P., Bohm, R., Mohamadzadeh, M., Ward, R.L., 2004. Defining the T-cell mediated responses in rotavirus-infected juvenile rhesus macaques. J. Virol. 78 (19), 10258 – 10264. Simpson, R., Aliyu, S., Iturriza-Gomara, M., Desselberger, U., Gray, J.,

2003. Infantile viral gastroenteritis: on the way to closing the diagnostic gap. J. Med. Virol. 70 (2), 258 – 262. Ushijima, H., Xin, K.Q., Nishimura, S., Morikawa, S., Abe, T., 1994. Detection and sequencing of rotavirus VP7 gene from human materials (stools, sera, cerebrospinal fluids, and throat swabs) by reverse transcription and PCR. J. Clin. Microbiol. 32 (12), 2893 – 2897. Ward, R.L., Bernstein, D.I., Smith, V.E., Sander, D.S., Shaw, A., Eiden, J.J., Heaton, P., Offit, P.A., Clark, H.F., 2004. Rotavirus immunoglobulin a responses stimulated by each of 3 doses of a quadrivalent human/bovine reassortant rotavirus vaccine. J. Infect. Dis. 189 (12), 2290 – 2293. Wilde, J., Eiden, J., Yolken, R., 1990. Removal of inhibitory substances from human fecal specimens for detection of group A rotaviruses by reverse transcriptase and polymerase chain reactions. J. Clin. Microbiol. 28 (6), 1300 – 1307. Xu, M., Miller, M.S., 2004. Determination of murine fetal Cyp1a1 and 1b1 expression by real-time fluorescence reverse transcription-polymerase chain reaction. Toxicol. Appl. Pharmacol. 201 (3), 295 – 302. Xu, L., Harbour, D., McCrae, M.A., 1990. The application of polymerase chain reaction to the detection of rotaviruses in faeces. J. Virol. Methods 27 (1), 29 – 37. Yamakawa, Y., Oka, H., Hori, S., Arai, T., Izumi, R., 1995. Detection of human parvovirus B19 DNA by nested polymerase chain reaction. Obstet. Gynecol. 86 (1), 126 – 129.