Materials and Reagents

1. For male genital secretion (GS) processing
   1. Gibco^® Hank’s balanced salt solution (HBSS) (no calcium, no magnesium, no phenol red) (Life Technologies, catalog number: 14175-095 )
   2. Gibco^® RPMI 1640 medium (Life Technologies, catalog number: 11875-093 )
   3. GemCell^TM U.S. origin fetal bovine serum (FBS) (Gemini-Bio Products, catalog number: 100-500 )
   4. Gibco^® penicillin-streptomycin (10,000 U/ml) (Life Technologies, catalog number: 15140-122 )
   5. Gibco^® L-Glutamine (200 mM) (Life Technologies, catalog number: 25030-024 )
   6. Nystatin suspension (10,000 units/ml in DPBS) (Sigma-Aldrich, catalog number: N1638 )
   7. Dimethyl sulfoxide (Hibri-Max^TM, sterile-filtered) (Sigma-Aldrich, catalog number: D2650 )
   8. Waxie^® bleach (Waxie Sanitary Supply, catalog number: 170016 )
   9. Viral transport medium (VTM) (see Recipes)
   10. 20% GS complete RPMI medium (see Recipes)
   11. 20% GS freeze medium (see Recipes)
       
       
2. For RNA and DNA extraction and cDNA generation
   1. QIAamp DNA mini kit (250 or 50 QIAamp mini spin columns) (QIAGEN, catalog numbers: 51306 or 51304 )
   2. High pure viral RNA kit (Roche Diagnostics, catalog number: 11858882001 )
   3. Invitrogen^TM SuperScript^® III first-strand synthesis system (Life Technologies, catalog number: 18080-051 )
      
   4. Seal-Rite 1.5 ml microcentrifuge (flip-top) tube (natural) (USA Scientific, catalog number: 1615-5500 )
      Note: These tubes are autoclaved before use.
      
   5. Micro tubes 1.5 ml (type D, without skirted base and with assembled neutral screw cap) (SARSTEDT AG, catalog number: 72.692.005 )
   6. Applied Biosystems^® MicroAmp^® reaction tube with cap (0.2 ml) (Life Technologies, catalog number: N8010540 )
   7. 1x Dulbecco’s phosphate buffered saline (DPBS) (Corning, Cellgro^TM, catalog number: 21-031-CV )
   8. EDTA (0.5 M pH 8.0, molecular biology grade) (Promega Corporation, catalog number: V4231 )
   9. Molecular biology grade sterile purified water (RNase, DNase, proteinase free) (Corning, Cellgro^TM, catalog number: 46-000-CM )
   10. Tris hydrochloride (1 M solution pH 8.0, molecular biology grade) (Thermo Fisher Scientific, catalog number: BP1758-500 )
       Note: 1 M Tris requires to be diluted to 10 mM with molecular grade water.
   11. Ethyl alcohol pure (200 proof molecular biology grade) (Sigma-Aldrich, catalog number: E7023 )
   12. MgCl₂ (50 mM solution) (Bio-Rad Laboratories, catalog number: 170-8872 )
       
       
3. For quantitative Real Time PCR (RT-qPCR)
   1. DNA plasmids or amplicons to be used as standards for RT-qPCR
      Note: Quantification standards for the different herpes viruses were obtained using DNA plasmid preparations with known concentrations. We provided the nucleotide sequences for each standard amplicon (in a separate fasta file from 5’ to 3’) (please see Supplementary Material). Oligonucleotides can be custom synthetized (e.g. Ultramer^® Oligonucleotides, Integrated DNA technologies [IDT]) and used directly as quantification standard, after measuring the amount of PCR-component template by spectrophotometry or droplet digital PCR.
      If desired, amplicons can also be inserted into a plasmid using standard kits (e.g. Zero Blunt^® TOPO^® PCR Cloning Kit, catalog number: K2800J10) and stocks of construct can be generated with large batch cultures (using standard protocols). After purification, quantify plasmid concentration by spectrophotometry (or ddPCR) and extract clone vectors using standard procedures. Consider linearization to increase efficiency and avoid supercoil formation.
   2. HIV RNA standard can be obtained from the DAIDS Virology Quality Assurance (VQA) Program (https://www.aidsreagent.org/, 150,000 copies HIV-1 RNA/ml spiked into negative plasma, catalog number: 3443 )
   3. Molecular biology grade sterile purified water (Corning, Cellgro^TM, catalog number: 46-000-CM)
   4. Primers and probes (Table 1)
      

Equipment

1. For male genital secretion (GS) processing
   1. Fisherbrand^™ polyethylene hinged-lid containers (Thermo Fisher Scientific, catalog number: 03-405-40 )
   2. Curwood Parafilm MTM laboratory wrapping film (4-inch W X 125-ft L) (Thermo Fisher Scientific, catalog number: 13-374-10 )
   3. Fisherbrand^™ Infecon^™ specimen transport bags (Thermo Fisher Scientific, catalog number: 19-287-215 )
   4. Corning^® 15 ml centrifuge tube (sterile) (Corning, catalog number: 430052 )
   5. Fisherbrand^TM sterile polystyrene disposable serological pipets with magnifier stripe for 1 ml, 2 ml, 5 ml, 10 ml (Thermo Fisher Scientific, catalog numbers: 13-676-10G , 13-675-3C , 13-676-10H , 13-676-10J )
   6. VWR^® disposable aspirating pipets (polystyrene, sterile) (VWR International, catalog number: 414004-264 )
   7. Micro tubes (2.0 ml, Type I, with skirted base and assembled neutral screw cap) (SARSTEDT AG, catalog number: 72.694.006 )
   8. 2.0 ml Ext FS CryoElite sterile cryogenic vials (yellow-cap) (Wheaton Science Products, catalog number: W985866 )
   9. Nalgene^® Mr. Frosty^TM freezing container (Thermo Fisher Scientific, catalog number: 5100-0001 )
   10. Allied^® 1,500 ml disposable collection canisters (Allied Healthcare Products, catalog number: 20-08-0003 )
   11. Sorvall^TM RC4 General Purpose Floor Model Centrifuge (Thermo Fisher Scientific, catalog number: 75004481 )
   12. Original Pipetman Aid^® Pipet Controller (Drummond Scientific Company, catalog number: 4-000-111-TC )
   13. Stericup-HV, 0.45 µm, PVDF (500 ml) (EMDMILLIPORE, catalog number: SCHVU05RE )
   14. Stericup-GV, 0.22 µm, PVDF (500 ml) (EMDMILLIPORE, catalog number: SCGVU05RE )
       
       
2. For RNA and DNA extraction and cDNA generation
   1. Fisher^® vortex genie 2, analog vortex mixer (Thermo Fisher Scientific, catalog number: 02-215-365 )
   2. VWR^® digital dry block heaters (or 2 heat blocks) (VWR Scientific Products, catalog number: 12621-088 )
   3. Allegra^® 64R high performance Benchtop centrifuge (Beckman Coulter, item number: 367585 )
   4. Eppendorf^® centrifuge (model: 5430 , 120 V, with Rotor, model: FA-45-30-11) (Eppendorf, 120V no longer available only 230V catalog number: 5427 000.216)
      
   5. Applied Biosystems^® GeneAmp^® PCR system 9700 (96-well gold-plated) (Life Technologies, catalog number: 4314878 )
      
      
3. For quantitative Real Time PCR (RT-qPCR)
   1. Applied Biosystems^® MicroAmp^® Optical 96-Well reaction plate (Life Technologies, catalog number: N8010560 )
   2. Applied Biosystems^® MicroAmp^® optical adhesive film (Life Technologies, catalog number: 4311971 )
   3. Applied Biosystems^® MicroAmp^® 96-Well Tray/Retainer Set (Life Technologies, catalog number: 403081 )
   4. Corning^® 50 ml centrifuge tube (sterile) (Corning, catalog number: 430290 )
   5. Applied Biosystems^® TaqMan^® environmental master mix 2.0 (200 reactions) (Life Technologies, catalog number: 4396838 )
   6. Applied Biosystems^® 7900HT fast Real-Time PCR system with Fast 96-Well block module (Life Technologies, catalog number: 4351405 )

Procedure