Qiaamp viral mini kit

Viral RNAs were extracted from 140 μl of virus-containing apical wash using a QIAamp viral mini kit from Qiagen, according to manufacturer’s protocol, and real-time PCR was performed using RNA Ultrasense One-Step qRT-PCR from Invitrogen targeting the N gene of HKU1. About 10 μl of viral RNA extract was mixed with 10 μl of master mix containing 2.3 μl of H₂O, 1 μl of enzyme, 4 μl of 5 × buffer, 0.2 μl of 10 μM probe (5′-TTGAAGGCTCAGGAAGGTCTGCTTCTAA-3′), 1 μl of 10 μM primer HKU1qPCR-F (5′-CTGGTACGATTTTGCCTCAA-3′), 1 μl of 10 μM primer HKU1qPCR-R (5′-ATTATTGGGTCCACGTGATTG-3′), and 0.5 μl of MgSO₄. Following an incubation at 42 °C for 5 min and a denaturation step of 5 min at 95 °C, 40 cycles of amplification were performed for 3 s at 95 °C and 30 s at 60 °C on a Roche Light Cycler 480 machine. A serially diluted synthetic DNA fragment containing the sequence from 28,849 to 28,949 of the HKU1 genome was used as the quantitative standard to calculate the viral genome copy number in the samples for real-time PCR.

Viral RNA was extracted from culture supernatants using the QIAamp Viral Mini Kit (Qiagen CA, USA) according to the manufacturer's instructions. The complete HN gene was amplified by RT-PCR using three sets of primers previously described [1 (link)].
The PCR amplicons were electrophoresed on a 2% agarose gel (Sigma-Aldrich Co., USA) stained in 2 μg/ml ethidium bromide (Sigma-Aldrich Co., USA) solution and visualized using the E-box gel documentation system (Vilber Lourmat, France). The amplicons were subsequently purified using Exonuclease I/Shrimp Alkaline Phosphatase (ExoSap-IT) enzyme (Affymetrix, USA) and sequenced on both strands on an automated 3500xL Genetic Analyzer (Applied Biosystems, USA) using the same primers. Cycle sequencing was performed using the Big Dye Terminator Cycle sequencing kit v3.1 (Applied Biosystems, USA).

Viral RNA was extracted from infected culture supernatants with a QIAamp Viral Mini Kit (Qiagen, Inc., USA), according to the manufacturer’s instructions. Partial VP1 gene (3′-end of VP1 gene) was amplified by RT-PCR using primers 292 (5′-MIGCIGYIGARACNGG-3′, position: 2612–2627) and 222 (5′-CICCIGGIGGIAYRWACAT-3′, position: 2969–2951) as previously described (Oberste et al. 2006 (link); Vignuzzi et al. 2005 (link)). PCR amplicons (~350 bp) were analyzed by electrophoresis using 1.0 % Agarose gels (Sigma-Aldrich Co., USA), stained in ethidium bromide (0.5 μg/ml) and visualized using the Alpha Imager (Alpha Innotech, USA). Amplicons were purified using Exonuclease I/Shrimp Alkaline Phosphatase (ExoSap-IT) enzyme (Affymetrix, USA) and sequenced directly in both directions using the RT-PCR primers. Sequencing was performed using Big Dye Terminator Cycle sequencing kit v3.1 (Applied Biosystems, USA) and analyzed using the automated 3500xL Genetic Analyzer (Applied Biosystems, USA) according to the manufacturer’s instructions.

Samples of duck feces were collected on duck cloaca using cotton swabs and conserved at −23 ℃ from ducks captured for marking within duck ecology and migration studies. The species sampled were Mallard Anas platyrhynchos, Pintail Anas acuta, and Teal Anas crecca. Captures were performed in São Jacinto Dunes Nature Reserve (Aveiro) and at EVOA (in Tagus River Estuary Nature Reserve, Vila Franca de Xira) since these are areas of high concentration of ducks where long-term duck ecology studies have been performed (see Figure 1). Ducks were visually marked with nasal saddles and could be followed on the field. A license to capture and mark ducks was obtained from Instituto da Conservação da Natureza e das Florestas (ICNF), Portugal (permit number 40/2021).
Fecal swabs were thoroughly mixed by vortexing in 500 µL of phosphate-buffered saline (PBS) pH 7.2. RNA was extracted from the fecal suspension using the QIAamp viral mini kit (Qiagen, Hilden, Germany) according to the manufacturer’s instructions using 140 µL of the clarified supernatants. Eluted RNA was then kept at −80 °C until further processing.

Assays were performed using real-time RT-PCR. RNA was extracted from the whole blood specimens using QIAamp Viral Mini Kit following the manufacturer’s protocol (QIAGEN GmbH, Hilden, Germany). Primers and probes targeting the G2 gene of the virus were then used to amplify a 94-nucleotide fragment using a TaqMan real-time RT-PCR assay. The PCR amplification of targeted viral sequence was performed in a 25-μL reaction mix containing 12.5 μL of 2× RT-PCR buffer,1 μL of 25× RT PCR enzyme, 0.25 μL of forward primer (40 µM), 0.25 μL of reverse primer (40 µM), 0.25 μL of probe (40 µM), and 5.75 μL of nuclease-free water. Five microliter of RNA template was then added to the reaction mix. An RVFV-positive control (RNA extracted from RVFV positive sample) and a negative control were included in the PCR reaction setup. Amplification was performed using Rotor-Gene^® Q real-time PCR system (QIAGEN GmbH), reverse transcription was performed at 50°C for 30 minutes followed by Taq activation at 95°C for 15 minutes, and then PCR was conducted for 45 cycles at 94°C for 15 seconds and 60°C for 1 minute. Following PCR analysis, any sample with a cycle threshold ≤ 37 was considered positive. The primers and probes used for the real-time PCR for this study are shown in the Annex.

Blood samples were collected in tubes with EDTA anticoagulant as the protocol of the Department of Surveillance, Prevention and Control of Sexually Transmitted Infections, HIV/AIDS and Viral Hepatitis of the Brazilian Ministry of Health for investigation of virological failure in patients undergoing antiretroviral therapy in Brazil. Viral RNA was extracted from plasma using QIAamp Viral mini Kit (Qiagen, Hilden, Germany). The protease (PR) and part of the reverse transcriptase (RT) sequencing of the HIV-1 polymerase gene (pol) was performed using the ViroSeqTM HIV-1 Genotyping System (Abbott Laboratories, US) and TRUGENE^® HIV-1 Genotyping Assay (Siemens Diagnostics, US) and analyzed using the ABI PRISM 3100 automatic DNA sequencer (Applied Biosystems, US) and OpenGene^® Sequencing System (Siemens Diagnostics, US), respectively. Genbank accession numbers: MN971800-MN972432.