Patients and Samples

During December 2011, we studied 301 randomly selected persons living in central Italy. Most (251) were diseased patients whose blood samples had been submitted to the Virology Unit, Pisa University Hospital (Pisa, Italy), by local hospitals for routine virologic analysis; the remaining 50 were healthy blood donors. The patients comprised 151 HIV-infected persons (mean ± SD age 47 ± 14 years [range 18–80 years]; 115 men) who, before initiation of the study, had received no antiretroviral treatment. The patients also were examined for xenotropic murine-leukemia virus-related virus in a previous study (14). The other 100 patients (mean ± SD age 56 ± 8 years [range 36–69 years]; 71 men) were solid organ transplant recipients: 50 had received a liver transplant, and 50 had received a kidney transplant. Plasma samples were collected from patients on the day of transplant and then at selected times after transplant. Aliquots were prepared immediately, stored, and kept under sterile conditions at –80°C until use. Written informed consent was collected from each patient.

HGyV DNA Detection

Viral DNA was extracted from 400 µL of peripheral whole blood or 200 µL of plasma by using the Maxwell 16 System (Promega, Madison, WI, USA) or QIAamp DNA blood kit (QIAGEN, Hilden, Germany), respectively, according to the manufacturers’ instructions. Extracted DNA was amplified with 2 PCR protocols (developed and provided by V. Sauvage et al.), which target the VP1 gene of the viral genome.

The first amplification was a single-step TaqMan real-time PCR (rtPCR) designed on a 72-nt fragment. The assay was performed in a 25-µL volume containing 400 nmol/L of each primer (HGyV-rtFP: 5′-TGCTTGCAACAATGCCTTTAGA-3′; HGyV-rtRP: 5′-CCCTGCAAGTGCTGAGGATAA-3′), 200 nmol/L double-labeled probe (HGyV-rtP: 5′-FAM-CAAAGAGCAAAATCGGAGGCCCTAACC-TAMRA-3′) 5 µL extracted viral DNA, and the Universal PCR Master Mix (Applied Biosystems, Foster City, CA, USA) containing deoxynucleoside triphosphate, and Taq DNA polymerase. Reaction was run in triplicate for each sample in an iQCYCLER rtPCR detection instrument (Bio-Rad Laboratories, Hercules, CA, USA) by using a standardized program (95°C 10 min; 45 cycles of 15 s at 95°C, and 60°C 60 s; and 40°C 30 s). A sample was considered rtPCR positive when HGyV DNA was detected in 2 of 3 replicas and when amplifications were specific as determined by 2% agarose gel electrophoresis.

A 178-bp fragment was amplified by a nested PCR format, described by Sauvage et al. (1) with modifications. This PCR was performed for 25 cycles with sense primer HGyV-OF (5′-CAAAATCGGAGGCCCTAACCC-3′) and antisense primer HGyV-OR (5′-ATGCCTGAATAGCTGCCAGCC-3′) under the following conditions: denaturation at 94°C for 60 s, annealing at 55°C for 60 s, and extension at 72°C for 45 s. The product of this reaction (10 µL) was then re-amplified for 35 cycles with internal primers HGyV-IF (5′-GGTCAGCACAAACGACGCAG-3′) and HGyV-IR (5′- AGGTCTCCCATAGCGTCCAG-3′) at the same PCR conditions. The reactions were conducted in a 50-µL PCR mixture containing Taq DNA polymerase, each deoxynucleoside triphosphate at a concentration of 10 mmol/L, primers (20 µmol/L each), and optimized buffer components.

All samples were tested at least in duplicate and on different occasions. The amplified product was analyzed by electrophoresis on a 2% agarose gel after ethidium bromide staining. Amplicon size was compared with standard molecular mass markers. To minimize contamination risk, serum handling, DNA extraction, PCR amplification, and electrophoresis analysis were conducted in separated rooms. Negative controls were added during DNA extraction and PCR amplification. To validate the amplification process, positive controls (obtained from M.E.) were run in each PCR.

Sequence Analysis

All HGyV PCR–positive isolates were characterized by sequencing a 138-bp fragment (from nt 1328 to 1465 of VP1 gene of the representative isolate FR823283) encompassing the target region of nested PCR. PCR amplicons, purified from the gel by using a QIAquick Gel Extraction Kit (QIAGEN, Chatsworth, CA, USA), were sequenced by using the Big Dye Terminator Cycle Sequencing Kit (Applied Biosystems) and an automatic DNA sequencer (ABI model 3130; Applied Biosystems). Nucleotide sequences were aligned with the only sequence available at GenBank at the time of writing and by using the ClustalW algorithm included in BioEdit version 7.0.9.0 (www.clustal.org).

Evidence of HGyV Infections

No samples from 50 healthy blood donors studied yielded positive results for HGyV (Table 1). Of 4 samples in which HGyV DNA was detected, 3 (6%) were from kidney transplant recipients and 1 (0.7%) was from an HIV-infected patient.

Longitudinal Study of HGyV Viremia in Transplant Recipients

We examined plasma samples from 100 transplant patients for whom we had sequential samples obtained at selected times after transplant. Three of these patients who had received a kidney transplant tested positive for HGyV DNA, indicating that they had systemic HGyV infection. When additional samples of these patients were examined, a similar pattern emerged (Table 2). Plasma samples from 2 transplant recipients were already HGyV positive when they were first examined before transplantation. Subsequently, HGyV DNA detection was intermittent in the posttransplant samples: it was positive at month 12 (patient AL) and 6 (patient CV) but negative in the other samples tested. For patient MG, we examined 4 blood samples obtained before and after HGyV detection in plasma. At all these times, the plasma tested repetitively negative for HGyV DNA.

Genetic Analysis of HGyV Isolates

Figure A1

Figure A1. . . Alignment of the nucleotide sequences of the human gyrovirus genome on the basis of a 138-bp segment (nt 1328–1465) of the viral protein 1 gene. The sequences of the...

Sequencing was conducted on all 6 PCR fragments obtained. All the isolates were related to the previously published strain, and the sequences obtained were virtually identical in the nucleotide fragment examined (Figure A1). When blood specimens from the same patient were sequenced, no nucleotide change was noted among the viral sequence fragments obtained at any time.