Ethics Statement

This study was conducted in accordance with the Declaration of Helsinki and with protocol code no. 40z, Register of Non-Covid Trials 2022. The study was approved by the Ethical Committee of the Lazzaro Spallanzani Institute MpoxCohort protocol “Studio di coorte osservazionale monocentrica su soggetti che afferiscono per sospetto clinico o epidemiologico di malattia del vaiolo delle scimmie (mpox).”

In Vitro Experiments

We maintained Vero E6 cells in modified eagle medium supplemented with 10% heat-inactivated fetal calf serum (FCS) at 37°C in a humidified atmosphere of 5% CO₂ and exposed to MPXV isolate hMpxv/Italy/un-INMI-Pt2/2022, clade/lineage IIb B.1 (GISAID accession no. EPI_ISL_13251120 [https://www.gisaid.org]; GenBank accession no. ON745215.1) for 1 hour and 30 minutes at 37°C at a multiplicity of infection of 0.01. At the end of the adsorption period, we washed and incubated cells at 37°C; at 30 minutes and 1, 2, 3, 4, 6, 24, and 48 hours postinfection (hpi), we harvested, inactivated, and tested cells and supernatants for MPXV DNA and G2R-mRNA presence by digital droplet PCR (ddPCR).

Clinical Samples

During May–September 2022, a total of 29 samples (7 nasopharyngeal swab, 10 skin lesion swab, 8 semen, and 4 urine samples) were collected for diagnostic purposes from 15 patients admitted to the National Institute for Infectious Diseases (INMI) Lazzaro Spallanzani in Rome, Italy. Patients had a positive diagnosis of mpox within 7 days of symptom onset. We retrospectively analyzed those samples.

MPXV DNA and G2R-mRNA Quantification

To establish the presence of viral DNA, we first analyzed samples from different anatomic sites of patients with an mpox diagnosis by a commercial MPXV real-time PCR kit (Jiangsu BioPerfectus Technologies Co., Ltd., http://www.bioperfectus.com) on the ELITe InGenius instrument (ELITechGroup SAS, https://www.elitechgroup.com), obtaining a semiquantitative measure by cycle threshold (Ct) value. We further analyzed biological samples that tested positive for MPXV DNA by real-time PCR and cell supernatants from the in vitro experiments by using ddPCR to obtain quantitative results expressed as copies per milliliter. In brief, we extracted 140 μL of supernatant from infected cells and biologic samples by using the QIAamp Viral DNA Mini Kit (QIAGEN, https://www.qiagen.com) according to the manufacturer’s instructions. We quantified MPXV DNA by using the QX200 AutoDG Digital Droplet PCR system (Bio-Rad Laboratories, https://www.bio-rad.com), as previously described (9).

To perform G2R-mRNA quantification, we extracted total RNA by using the RNeasy Mini Kit (QIAGEN) according to the manufacturer’s instructions. To selectively degrade any traces of DNA, we treated RNA extracted with DNase (TURBO DNase Kit; Thermo Fisher Scientific, https://www.thermofisher.com). We then reverse transcribed 10 μL of RNA per sample according to the instructions of the SuperScript IV First-Strand cDNA Synthesis reaction kit (Thermo Fisher) by using 50 μmol OLigo d(T)20 as primers to select mRNA. We performed quantification by using the Bio-Rad QX200 AutoDG ddPCR system, targeting the early gene, G2R-mRNA (10). To confirm the absence of MPXV DNA fragments, all RNA-extracted samples underwent MPXV DNA PCR after treatment with DNase.

Statistical Analysis

We performed linear regression analysis by using GraphPad Prism version 9 (https://www.graphpad.com). We expressed results as correlation coefficients (r).

In Vitro Cell Culture Experiments

Figure 1

Figure 1. In vitro kinetics of MPXV infection in Vero E6 cell line in study of role of direct sexual contact in human transmission of MPXV, Italy. Cell-associated G2R-mRNA, a surrogate marker...

To establish if G2R-mRNA could be considered a surrogate marker of ongoing MPXV replication, we tested for its presence in vitro in infected Vero E6 cells. Cell-associated G2R-mRNA was detected at low levels until 30 minutes postinfection, when it started to increase, showing a peak at 1 hpi (Figure 1). This result was expected because the G2R gene is early transcribed during viral infection. After 1 hpi, we observed a slight increase in cell-associated G2R-mRNAs throughout the infection until 24 hpi; levels remained stable thereafter. All RNA samples treated with DNase were negative for MPXV DNA, confirming the absence of MPXV DNA fragments and the strength of the setup method (data not shown).

MPXV DNA levels released in the supernatant steadily increased at each timepoint, peaking at 24 hpi. We observed substantially lower levels of DNA released in the supernatants for mRNA cell-associated DNA, starting at 30 minutes postinfection and continuing throughout the observation period.

MPXV DNA and G2R-mRNA in Clinical Specimens

Figure 2

Figure 2. Linear regression analysis of Ct values and MPXV DNA levels in clinical specimens from study of role of direct sexual contact in human transmission of MPXV, Italy. Results show highly...

MPXV DNA and G2R-mRNA were present in NPS, skin lesion swabs, urine, and semen samples (Table). All samples were positive for MPXV DNA by diagnostic real-time PCR (mean Ct 27.3, range 13.3–37.7), except for all urine and 2 semen samples that showed negative results (Ct >45.0). We performed absolute quantification of positive MPXV DNA samples by ddPCR, and linear regression analysis found a highly negative correlation (r = −0.99) between the Ct values and the amount of MPXV DNA (Figure 2).

G2R-mRNA was detectable in all skin lesions (r = 0.71) and 4 out of 7 NPS samples (r = 0.64), showing a good correlation with MPXV DNA in both matrices. Of note, we observed low positivity for G2R-mRNA presence in only 2 of 8 semen samples; therefore, we could not correlate those results with DNA levels. Nevertheless, we emphasize that semen samples with the highest DNA copy numbers tested negative for G2R-mRNA, which is different from what we observed in other matrices (Table).